Whole-body hyperthermia-induced thermotolerance is associated with the induction of Heat Shock Protein 70 in mice
Cardiovascular Research Center, Department of Comparative Medicine, Pig Research Institute Taiwan, PO Box 23, Chunan, Miaoli 35099, Taiwan, Republic of China
*Author for correspondence (e-mail: wen-chuan{at}mailcity.com)
Accepted 30 October 2001
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Summary |
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Key words: whole-body thermotolerance, heat shock protein 70, HSP70, antioxidant enzyme, preconditioning, mouse.
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Introduction |
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Heat shock proteins (HSPs), recognized as molecular chaperones, are families of highly conservative stress proteins (Lindquist and Craig, 1988; Ellis and van der Vies, 1991
; Hutter et al., 1996
). The 70 kDa HSP family (HSP70) is categorized into constitutive and inducible forms (Lindquist and Craig, 1988
), which contribute to stress tolerance by increasing the chaperone activity in the cytoplasm (Nollen et al., 1999
). The inducible form of HSP70 (HSP70i) has been proposed as a predictor or indicator for thermotolerance at either the cell or animal level (Li and Mak, 1989
; Flanagan et al., 1995
). Intriguingly, the protective role of HSP70i in mammalian thermal death has yet to be determined.
Reactive oxygen species (ROS), which are postulated to be cellular toxicants (Gorman et al., 1999; Davidson et al., 1996
), can be induced through hyperthermia (Flanagan et al., 1998
). Moreover, it has been proposed that increased superoxide dismutase (SOD) activity contributes to cellular thermotolerance (Loven et al., 1985
). Several studies have revealed that ROS induces HSP synthesis, which is critical for cellular thermotolerance development (Gorman et al., 1999
; Wong et al., 1998
; Ciacarra et al., 1994
), but the role of antioxidant enzymes in WBT is controversial (Currie and Tanguay, 1991
; Stears and Yellon, 1994
; Joyeux et al., 1997
), and their role in mouse WBT remains to be determined further. The aim of this study was to explore the roles of HSP70i and the activities of antioxidant enzymes in mouse WBT.
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Materials and methods |
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Whole-body hyperthermia
Prior to the treatments, the animals were stabilized at room temperature (25±1°C) for 7 days. Whole-body hyperthermia (WBH) was performed on a heating pad. Animals were anesthetized and maintained throughout the operation by injection of 2.5 % Avertin saline (20 µl g1 body mass, Aldrich, USA). A 100 % stock of Avertin contains 1 g 2,2,2-tribromoethyl alcohol in 1 ml tert-amyl alcohol. The animals were taped onto the heating pad and a rectal thermostat probe was inserted (Harvard, USA). The temperature was maintained at 41±0.1°C. To prevent any unnecessary heat injury to the cephalic organs, a cushion was placed under the head. During the operation, room temperature was maintained at 25±1°C. The sham control group received Avertin treatment but no preheat treatment. Preconditioning treatments were administered for intervals of 15, 30 and 45 min. Following a 48 h recovery period, a lethal challenging dose (41°C, for 60 min) was given. The survival rate (SR) of each treatment is defined as the number of animals surviving after hyperthermia challenge / number of animals before hyperthermia challenge. Whole-body thermotolerance (WBT) of animals was assessed with protection index (PI) defined as the SR of preconditioned animals after challenge / SR of non-preconditioned animals after challenge.
Gel electrophoresis and immunoblot analysis
To avoid blood contamination, liver tissues from both the control and heat-treated ICR mice were thoroughly washed with 0.9 % saline. Homogenization of the tissues was performed by a polytron (PT3100, Switzerland) in homogenization buffer (10 g sucrose, 4.0 mg pefablo SC, 0.5 mol l1 Tris-HCl, pH 6.8, in 100 ml). The crude homogenates were then centrifuged at 12,000 g for 5 min at 4°C (Kubota1720, Japan). The supernatants were collected for further experiments.
One-dimensional polyacrylamide gel electrophoresis (PAGE) and the immunoblot analysis were conducted as described previously (Lee et al., 1996). The supernatant of the tissue homogenate was lysed in a sample buffer (pH 6.8) containing 62.5 mmol l1 Tris-HCl, 2 % sodium dodecylsufate (SDS), 5 % 2-mercaptoethanol, 10 % glycerol and 0.002 % Bromophenol Blue. The sample was boiled for 5 min, cooled in an ice bath and then centrifuged at 12,000 g for 3 min. Using bovine serum albumin as a standard, the protein concentration was determined (Lowry et al., 1951
). Approximately 75 µg of liver samples were subjected to 9 % SDS-PAGE.
For immunoblotting analysis, protein bands in the slab gels were transferred to a nitrocellulose membrane (Hybond-C extra, Amersham, USA) by a semi-dry method (OWL Scientific Plastics, Cambridge, UK). The blot was incubated for 1 h in a 3 % gelatin solution (pH 7.4) containing 20 mmol l1 Tris-HCl, 500 mmol l1 NaCl, 0.05 % Tween 20 (TTBS) and then rinsed with TTBS. Subsequently, the membrane was incubated with rabbit anti-human HSP70 (Hsp72) polyclonal antibody (SPA-812, StressGen, Canada; diluted 1:1000 in TTBS containing 1 % gelatin) and against porcine HSP90 (diluted 1:1000 in TTBS containing 1 % gelatin) (Huang et al., 1999) for 1 h at room temperature. After three washes in TTBS, the membrane was reacted with goat-anti-rabbit antibody conjugated with alkaline phosphate (Sigma, USA; diluted 1:5000 in TTBS containing 1 % gelatin) for 1 h at room temperature. The membrane was rinsed three times with TTBS and developed within 3 min by an alkaline phosphate conjugate substrate kit (BioRad, USA) at room temperature. For further quantitative analysis, gel and immunoblot images were obtained using a densitometer equipped with ImageQuant (Molecular Dynamics, USA).
Two-dimensional (2D)-PAGE was performed as described previously (King et al., 2000). Approximately 300 µg of protein was loaded onto the isoelectrofocusing (IEF) gel and electrophoresized at 400 V for 16 h and then at 800 V for 1 h. Subsequently, the IEF gels were laid onto 9 % SDS-polyacrylamide slab gels with a 4.75 % stacking gel in the second dimension. The immunoblotting methods and gel imaging assessment were as described above.
Activity assays of antioxidant enzymes
The methods were conducted as described previously (Lin et al., 1997). Tissues were sliced into small pieces and thoroughly washed with a 50 mmol l1 potassium phosphate buffer. Tissue homogenization was performed as described above and the supernatants were prepared for enzyme activity assays. Protein concentration was determined by the Lowry method (Lowry et al., 1951
). Total superoxide dismutase (SOD) activity was determined by means of inhibition of pyrogallol autooxidation (Marklund and Marklund, 1974
). At 420 nm, the optical density of the mixture was measured using a spectrophotometer (DU7500, Beckman, USA) at 25°C for 5 min.
A420 values, which ranged from 0.12 to 0.35, were recorded by a constant, per minute relationship increase under normal conditions. Catalase (CAT) activity was obtained spectrophotometrically by measuring H2O2 decomposition at 25°C and at 240 nm (Aebi, 1983
). The absorbancy decrease was recorded for 1 min:
A240/
t=15 s1 values ranged between 0.02 and 0.10. Glutathione peroxidase (GSPx) activity was assessed by the Flohe and Gunzler (1984
) method with minor modification. To inhibit CAT activity, tert-butyl hydroperoxide was employed as a substrate, rather than H2O2, and 1 mmol l1 sodium azide. Reaction rate was determined at 340 nm and 37°C for 5 min. All enzyme activities are expressed in unit mg1 of protein. One SOD activity unit is the prescribed amount of enzyme required to inhibit pyrogallol autooxidation by 50 %. 1 CAT unit decomposes 1 µmol of H2O2 min1 at 25°C. 1 GSPx unit results in 1 µmol of oxidized glutathione (GSH) min1.
Statistical analysis
The SAS GLM procedure was employed to analyze quantitative data (SAS Institute, 1989). Differences among groups were determined by the Duncan method. P values less than 0.05 were considered statistically significant.
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Results |
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Discussion |
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However, to confer WBT, molecules other than HSP70 or stress proteins may also perform a function via different mechanisms. A cellular study has indicated that, after heat exposure, SOD activity increased (Loven et al., 1985). It is assumed that this increase coincided with HSP synthesis, as well as the thermotolerance development, which therefore suggests that SOD protects cells from heat stress (Gorman et al., 1999
). Moreover, HL-60 cells are partially protected from hyperthermia-induced apoptosis, while incubated with exogenous CAT during heat exposure (Bicher, 1980
). Furthermore, overexpressed GSPx in human MCF-7 cells increases anti-oxidative stress ability (Doroshow, 1995
). Due to the compensation of other antioxidant enzymes, GSPx-deficient mice continue to survive normally (Ho et al., 1997
). However, data presented here show that the residual activities of SOD, CAT and GSPx are not altered, whereas that of HSP70i is (Table 3). The anti-heat-stress effect of antioxidant enzymes on WBT therefore requires further investigation.
In this study we have clearly demonstrated that, in mice, thermotolerance is substantially induced by WBH. Moreover, we have demonstrated that the levels of HSP70i in these mice can be increased. We did not find any contribution of antioxidant enzymes in this animal model and, therefore, we conclude that, in mice, WBT increased by WBH is associated with HSP70i and not with CAT, SOD or GSPx activities, which enables the animal to survive an acute heat stress.
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Acknowledgments |
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References |
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