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Chronic stress reduces serum but not liver metallothionein response to acute stress
Chem..Biol. Interactions, 88 (1993) 1 - 5 Elsevier Scientific Publishers Ireland Ltd.
1
CHRONIC STRESS REDUCES SERUM BUT NOT LIVER METALLOTHIONEIN RESPONSE TO ACUTE STRESS
M. GIRALT, A. ARMARIO AND J. HIDALGO
Departamento de Biologia Celular y Fisiologia, Facultad de Ciencias, Universidad Autdnoma de Barcelona, Bellaterra, Barcelona (Spain) (Received October 23rd, 1992) (Revision received February 15th, 1993) (Accepted February 16th, 1993)
SUMMARY
Rats subjected to chronic immobilization stress showed a reduced serum metallothionein (MT) response to acute immobilization stress compared to nonchronically stressed rats. In contrast, liver MT response to acute immobilization stress was not influenced by previous chronic immobilization stress. These results suggest that serum MT levels are likely under endocrine regulation and that they do not reflect directly liver MT levels. Instead it appears that both MT pools are regulated differently. The fact that liver MT is resistant to adaptation to chronic stress may be related to its physiological function.
Key words: Serum and liver metallothionein -- Acute stress -- Chronic stress
INTRODUCTION
Metallothionein (MT) is a low molecular weight, cysteine- and heavy metal-rich protein. Initially thought to be involved in heavy metal detoxification [1], it seems more likely that MT is involved in Zn and Cu metabolism [2,3] and in the adaptation of the organism to stress [4- 7]. MT has been demonstrated to be a good free radical scavenger in vitro [8,9] and an antioxidant role for liver MT during stress has recently been suggested [10]. Although the source of serum MT is not precisely known, it seems that the liver is a good candidate [11]. Furthermore, serum MT levels reflect liver MT changes at .least in some circumstances [10,12-15]. However, it is not known Correspondence to: J. Hidalgo, Departamento de Biologia Celular y Fisiologla, Unidad de Fisiologia Animal, Facultad de Ciencias, Universidad AutSnoma de Barcelona, Bellaterra, Barcelona 08193, Spain. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
whether or not serum MT is regulated independently of liver MT concentration. To that end, we have studied the effect of previous chronic stress on the response of serum and liver MT to acute stress. MATERIALS AND METHODS
Adult male Sprague-Dawley rats (weighing about 370 g at the beginning of the experiment) were maintained in groups of four per cage under standard conditions (food and water ad libitum, lights on from 07.00 to 19.00 h) and were used one week after their arrival to the laboratory. Rats were randomly assigned to either control or chronic stress groups. Chronically stressed rats were subjected to 2.5 h of daily immobilization stress [10,15] in the morning for 13 days. The rats were then assigned to three experimental groups: (a) basal-rats remained undisturbed in the animal room and sacrificed 18 h later; (b) starvation-rats were deprived of food and water for 18 h and then sacrificed; and (c) immobilizationrats were subjected to immobilization stress for 18 h and then sacrificed. Immobilized rats had no access to food and water. Rats were killed by decapitation; the trunk blood was collected at 4°C and the serum stored at - 2 0 ° C and the livers immediately removed and stored at -90°C. Later, the livers were homogenized with ice-cold 10 mM Tris-HC1, pH 8.2, containing 0.25 M sucrose, 2 mM 2-mercaptoethanol, 1 mM phenyl methyl sulfonyl fluoride and 10 mM sodium azide in a Potter-Elvehjem. The homogenates were centrifuged at 50 000 x g (30 min, 4°C), and the supernatants were stored at -20°C. Serum and liver cytosol aliquots were lyophilized for transport to Syracuse University, where they were reconstituted with distilled water for MT radioimmunoassay (RIA) by one of the authors (JH) following the established RIA procedure of Dr. J.S. Garvey [16]. The intra-assay coefficient of variation of the RIA was 5%. Results were analyzed with the Student's t-test and with one-way analysis of variance (ANOVA). RESULTS
Fig. 1 shows serum and liver MT of all the experimental groups. Liver MT levels were significantly (P at least < 0.05) increased by starvation, and immobilization plus starvation further increased them (P < 0.1). Chronic immobilization stress did not alter liver MT response to any stressor. In contrast to liver MT, serum MT levels were not increased by starvation. However, serum MT level~ were strongly increased (P < 0.001) by acute immobilization stress. Chronic immobilization stress did not alter basal and starvation serum MT levels, but strongly reduced (P < 0.01) serum MT response to acute immobilization stress. DISCUSSION
As expected [10,15], acute immobilization stress strongly increased liver MT levels. Because immobilized rats had no access to food and water and starvation
SERUM
MT
10 m
E e-
5 control
o__[]/ LIVER
MT
20 O1
/
chronic stress
O) ="
10
0 basal starvation stress Fig. 1. Influence of previous chronic immobilization stress on serum and liver metallothionein (MT) response to acute immobilization stress. Because immobilized rats had no access to food and water, a group of rats deprived of food and water (starvation) was included. Serum MT: • P < 0.001 vs. basal and starved rats; * P < 0.01 vs. control rats. Liver MT: • P at least < 0.05 vs. basal rats; • P < 0.01 vs. starved rats.
will increase liver MT levels [17], a group of rats subjected to starvation alone was included. As expected [6,7,10], the effect of starvation alone on liver MT levels was lower than that of immobilization plus starvation, and therefore a specific stress effect was present. In no case were liver MT levels altered by previous chronic immobilization stress. In contrast to liver MT, serum MT levels were increased by immobilization stress but not by starvation, which is in agreement with previous reports [6,7,10]. This suggests that serum MT levels are not directly related to liver MT
levels. However, it could be argued that the increase of liver MT levels was just too low as to see an increase in serum MT levels. If this were the case (to pass a threshold), then serum MT levels should be the same in control and chronically stressed rats, since both groups showed similar liver MT levels. However, chronically stressed rats showed lower serum MT levels during acute stress than control rats in spite of the fact that there was no difference in their liver MT levels. Therefore, the mere concentration of MT in the liver does not appear to be responsible for the serum MT levels observed during acute stress. In agreement with this hypothesis, we have observed that rats subjected to immobilization stress have higher serum MT levels than rats subjected to sham adrenalectomy eventhough the latter tended to have higher liver MT levels (data not shown). Thus, a mechanism independent of the liver MT content appears to be present in the regulation of serum MT levels. The fact that previous chronic immobilization stress reduced serum MT response to acute immobilization stress suggests that serum MT levels during stress are under endocrine control. A reduction of the response to an acute stress after chronic exposure to the same stressor is a typical feature of hormones such as glucocorticoids or catecholamines [18,19] and the two of them have been involved in serum MT regulation [15,20]. Although further work is needed, all these results suggest that the adaptation to chronic stress showed by serum MT is mediated by endocrine systems that become adapted to chronic stress. That previous chronic stress did not affect liver MT response to acute stress is interesting because it indicates that, in contrast to serum MT, liver MT is resistant to adaptation to stress. This could be important for the physiological function of this protein in the liver. It has recently been suggested that liver MT could function as an antioxidant during stress [10]. We have determined liver lipid peroxidation as measured by malondialdehyde formation caused by acute stress and found no differences between control and chronically stressed rats (data not shown). If MT is an antioxidant during stress in the liver, similar liver MT levels should be needed in control and chronically stressed rats during acute stress, since similar liver lipid peroxidation levels were observed. In contrast, no evidence was seen to support an antioxidant role for serum MT [10]. Instead, serum MT appeared to be involved in the metabolism of Zn, which is known to be influenced by endocrine systems [2]. This suggests a relationship between serum MT, Zn metabolism and endocrine systems, which merits further attention. In summation, the present results suggest that (a) the mere concentration of MT in the liver may not be responsible for serum MT levels, at least during acute stress, and (b) there appears to be an adaptation of serum but not liver MT to chronic stress, which may be related to the physiological function(s) of this protein. ACKNOWLEDGEMENTS
This study was supported in part by a grant of the FIS (90/0065-2-D) and of DGICYT (PB91-0489). The MT radioimmunoassays have been performed in the laboratory of Dr. Justine S. Garvey, Department of Biology, Syracuse University, Syracuse, New York, USA.
REFERENCES 1 M. Webb, Role of metallothionein in cadmium metabolism, in: B.C. Foulkes (Ed.), Handbook of experimental pharmacology, Springer Verlag, Berlin, 1986, pp. 281-337. 2 R.J. Cousins, Absorption, transport and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin, Physiol. Rev., 65 (1985) 238-309. 3 M.A. Dunn, T.L. Blalock and R.J. Cousins, Metallothionein, Proe. Soc. Exp. Biol. Med., 185 (1987) 107-199. 4 F.O. Brady, Synthesis of rat hepatic zinc thionein in response to the stress of sham operation, Life Sci., 28 (1981) 1647-1654. 5 S.H. Oh, J.T. Deagen, P.D. Whanger and P.H. Weswig, Biological function of metallothionein. V. Its induction by various stresses, Am. J. Physiol., 234 (1978) E282-E285. 6 J. Hidalgo, A. Armario, R. Flos and J.S. Garvey, Restraint stress induced changes in rat liver and serum metallothionein and in zinc metabolism, Experientia (Basel), 42 (1986a) 1006-1010. 7 J. Hidalgo, A. Armario, R. F|os, A. Dingman and J.S. Garvey, The influence of restraint stress in rats on metallothionein production and corticosterone and glucagon secretion, Life Sci., 39 (1986b) 611-616. 8 P.J. Thornalley and M. Vag~k, Possible role for mctallothionein in protection against radiation induced oxidative stress. Kinetics and mechanism of its reaction with superoxid¢ and hydroxyl radicals, Biochim. Biophys. Acta, 827 (1985) 36-44. 9 J.P. Thomas, G.J. Bachowski and A.W. Girotti, Inhibition of cell membrane lipid pcroxidation by cadmium- and zinc-metallothioneins, Biochim. Biophys. Acta, 884 (1986) 448-461. l0 J. Hidalgo, L. Campmany, M. Borr~s, J.S. Garvcy and A. Armario, Metallothioncin response to stress in rats: role in free radical scavening, Am. J. Physiol., 255 (1988a) E518-E524. I 1 K.G. Danielson, S. Ohi and P.C. Huang, Immunochemical detection of metallothionein in specific epitelial cells of rat organs, Proc. Natl. Acad. Sci., USA 79 (1982) 2301-2304. 12 I. Brcmner, J.N. Morrison, A.M. Wood and J.R. Arthur, Effects of changes in dietary zinc, copper and selenium supply and of endotoxin administration on metallothioncin I concentrations in blood cells and urine in the rat, J. Nutr., ll7 (1987) 1595-1602. 13 J.N. Morrison and I. Bremncr, Effect of maternal zinc supply on blood and tisucs mctallothioncin I concentrations in suckling rats, J. Nutr., ll7 (1987) 1588-1594. 14 M. Sato, R.K. Mehra and I. Brcmner, Measurement of plasma metallothioncin-I in assessment of zinc status of zinc-deficient and stressed rat, J. Nutr., ll4, (1984) 1683-1689. 15 J. Hidalgo, M. Giralt, J.S. Garvcy and A. Armario, Physiological role of glucocorticoids on rat serum and liver metallothionein in basal and stress conditions, Am. J. Physiol., 254 (1988b) E71-E78. 16 J.S Garvey, Metallothionein: structure/antigenicity and detection/quantitation in normal physiological fluids, Environ. Health Perspect., 54 (1984) 117-127. 17 I. Bremner and N.T. Davies, The induction of metallothionein in rat liver by zinc injection and restriction of food intake, Biochem. J., 149 (1975) 733-738. 18 A. Armario, A. Lopez-Calder6n, T. Jolin and J. Balasch, Response of anterior pituitary hormones to chronic stress. The specificity of adaptation, Neurosci. Biobehav. Rev., 10 (1986) 245-250. 19 E.A. Stone and R. McCarty, Adaptation to stress: tyrosine hydroxylase activity and catecholamines release, Neurosci. Biobehav. Rev., 7 (1983) 29-34. 20 J. Hidalgo, J.S. Garvey and A. Armario, The role of catecholamines and glucagon on serum and liver metallothionein response to restraint stress, Rev. Esp. Fisiol., 43 (1987) 433-438.
1
CHRONIC STRESS REDUCES SERUM BUT NOT LIVER METALLOTHIONEIN RESPONSE TO ACUTE STRESS
M. GIRALT, A. ARMARIO AND J. HIDALGO
Departamento de Biologia Celular y Fisiologia, Facultad de Ciencias, Universidad Autdnoma de Barcelona, Bellaterra, Barcelona (Spain) (Received October 23rd, 1992) (Revision received February 15th, 1993) (Accepted February 16th, 1993)
SUMMARY
Rats subjected to chronic immobilization stress showed a reduced serum metallothionein (MT) response to acute immobilization stress compared to nonchronically stressed rats. In contrast, liver MT response to acute immobilization stress was not influenced by previous chronic immobilization stress. These results suggest that serum MT levels are likely under endocrine regulation and that they do not reflect directly liver MT levels. Instead it appears that both MT pools are regulated differently. The fact that liver MT is resistant to adaptation to chronic stress may be related to its physiological function.
Key words: Serum and liver metallothionein -- Acute stress -- Chronic stress
INTRODUCTION
Metallothionein (MT) is a low molecular weight, cysteine- and heavy metal-rich protein. Initially thought to be involved in heavy metal detoxification [1], it seems more likely that MT is involved in Zn and Cu metabolism [2,3] and in the adaptation of the organism to stress [4- 7]. MT has been demonstrated to be a good free radical scavenger in vitro [8,9] and an antioxidant role for liver MT during stress has recently been suggested [10]. Although the source of serum MT is not precisely known, it seems that the liver is a good candidate [11]. Furthermore, serum MT levels reflect liver MT changes at .least in some circumstances [10,12-15]. However, it is not known Correspondence to: J. Hidalgo, Departamento de Biologia Celular y Fisiologla, Unidad de Fisiologia Animal, Facultad de Ciencias, Universidad AutSnoma de Barcelona, Bellaterra, Barcelona 08193, Spain. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
whether or not serum MT is regulated independently of liver MT concentration. To that end, we have studied the effect of previous chronic stress on the response of serum and liver MT to acute stress. MATERIALS AND METHODS
Adult male Sprague-Dawley rats (weighing about 370 g at the beginning of the experiment) were maintained in groups of four per cage under standard conditions (food and water ad libitum, lights on from 07.00 to 19.00 h) and were used one week after their arrival to the laboratory. Rats were randomly assigned to either control or chronic stress groups. Chronically stressed rats were subjected to 2.5 h of daily immobilization stress [10,15] in the morning for 13 days. The rats were then assigned to three experimental groups: (a) basal-rats remained undisturbed in the animal room and sacrificed 18 h later; (b) starvation-rats were deprived of food and water for 18 h and then sacrificed; and (c) immobilizationrats were subjected to immobilization stress for 18 h and then sacrificed. Immobilized rats had no access to food and water. Rats were killed by decapitation; the trunk blood was collected at 4°C and the serum stored at - 2 0 ° C and the livers immediately removed and stored at -90°C. Later, the livers were homogenized with ice-cold 10 mM Tris-HC1, pH 8.2, containing 0.25 M sucrose, 2 mM 2-mercaptoethanol, 1 mM phenyl methyl sulfonyl fluoride and 10 mM sodium azide in a Potter-Elvehjem. The homogenates were centrifuged at 50 000 x g (30 min, 4°C), and the supernatants were stored at -20°C. Serum and liver cytosol aliquots were lyophilized for transport to Syracuse University, where they were reconstituted with distilled water for MT radioimmunoassay (RIA) by one of the authors (JH) following the established RIA procedure of Dr. J.S. Garvey [16]. The intra-assay coefficient of variation of the RIA was 5%. Results were analyzed with the Student's t-test and with one-way analysis of variance (ANOVA). RESULTS
Fig. 1 shows serum and liver MT of all the experimental groups. Liver MT levels were significantly (P at least < 0.05) increased by starvation, and immobilization plus starvation further increased them (P < 0.1). Chronic immobilization stress did not alter liver MT response to any stressor. In contrast to liver MT, serum MT levels were not increased by starvation. However, serum MT level~ were strongly increased (P < 0.001) by acute immobilization stress. Chronic immobilization stress did not alter basal and starvation serum MT levels, but strongly reduced (P < 0.01) serum MT response to acute immobilization stress. DISCUSSION
As expected [10,15], acute immobilization stress strongly increased liver MT levels. Because immobilized rats had no access to food and water and starvation
SERUM
MT
10 m
E e-
5 control
o__[]/ LIVER
MT
20 O1
/
chronic stress
O) ="
10
0 basal starvation stress Fig. 1. Influence of previous chronic immobilization stress on serum and liver metallothionein (MT) response to acute immobilization stress. Because immobilized rats had no access to food and water, a group of rats deprived of food and water (starvation) was included. Serum MT: • P < 0.001 vs. basal and starved rats; * P < 0.01 vs. control rats. Liver MT: • P at least < 0.05 vs. basal rats; • P < 0.01 vs. starved rats.
will increase liver MT levels [17], a group of rats subjected to starvation alone was included. As expected [6,7,10], the effect of starvation alone on liver MT levels was lower than that of immobilization plus starvation, and therefore a specific stress effect was present. In no case were liver MT levels altered by previous chronic immobilization stress. In contrast to liver MT, serum MT levels were increased by immobilization stress but not by starvation, which is in agreement with previous reports [6,7,10]. This suggests that serum MT levels are not directly related to liver MT
levels. However, it could be argued that the increase of liver MT levels was just too low as to see an increase in serum MT levels. If this were the case (to pass a threshold), then serum MT levels should be the same in control and chronically stressed rats, since both groups showed similar liver MT levels. However, chronically stressed rats showed lower serum MT levels during acute stress than control rats in spite of the fact that there was no difference in their liver MT levels. Therefore, the mere concentration of MT in the liver does not appear to be responsible for the serum MT levels observed during acute stress. In agreement with this hypothesis, we have observed that rats subjected to immobilization stress have higher serum MT levels than rats subjected to sham adrenalectomy eventhough the latter tended to have higher liver MT levels (data not shown). Thus, a mechanism independent of the liver MT content appears to be present in the regulation of serum MT levels. The fact that previous chronic immobilization stress reduced serum MT response to acute immobilization stress suggests that serum MT levels during stress are under endocrine control. A reduction of the response to an acute stress after chronic exposure to the same stressor is a typical feature of hormones such as glucocorticoids or catecholamines [18,19] and the two of them have been involved in serum MT regulation [15,20]. Although further work is needed, all these results suggest that the adaptation to chronic stress showed by serum MT is mediated by endocrine systems that become adapted to chronic stress. That previous chronic stress did not affect liver MT response to acute stress is interesting because it indicates that, in contrast to serum MT, liver MT is resistant to adaptation to stress. This could be important for the physiological function of this protein in the liver. It has recently been suggested that liver MT could function as an antioxidant during stress [10]. We have determined liver lipid peroxidation as measured by malondialdehyde formation caused by acute stress and found no differences between control and chronically stressed rats (data not shown). If MT is an antioxidant during stress in the liver, similar liver MT levels should be needed in control and chronically stressed rats during acute stress, since similar liver lipid peroxidation levels were observed. In contrast, no evidence was seen to support an antioxidant role for serum MT [10]. Instead, serum MT appeared to be involved in the metabolism of Zn, which is known to be influenced by endocrine systems [2]. This suggests a relationship between serum MT, Zn metabolism and endocrine systems, which merits further attention. In summation, the present results suggest that (a) the mere concentration of MT in the liver may not be responsible for serum MT levels, at least during acute stress, and (b) there appears to be an adaptation of serum but not liver MT to chronic stress, which may be related to the physiological function(s) of this protein. ACKNOWLEDGEMENTS
This study was supported in part by a grant of the FIS (90/0065-2-D) and of DGICYT (PB91-0489). The MT radioimmunoassays have been performed in the laboratory of Dr. Justine S. Garvey, Department of Biology, Syracuse University, Syracuse, New York, USA.
REFERENCES 1 M. Webb, Role of metallothionein in cadmium metabolism, in: B.C. Foulkes (Ed.), Handbook of experimental pharmacology, Springer Verlag, Berlin, 1986, pp. 281-337. 2 R.J. Cousins, Absorption, transport and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin, Physiol. Rev., 65 (1985) 238-309. 3 M.A. Dunn, T.L. Blalock and R.J. Cousins, Metallothionein, Proe. Soc. Exp. Biol. Med., 185 (1987) 107-199. 4 F.O. Brady, Synthesis of rat hepatic zinc thionein in response to the stress of sham operation, Life Sci., 28 (1981) 1647-1654. 5 S.H. Oh, J.T. Deagen, P.D. Whanger and P.H. Weswig, Biological function of metallothionein. V. Its induction by various stresses, Am. J. Physiol., 234 (1978) E282-E285. 6 J. Hidalgo, A. Armario, R. Flos and J.S. Garvey, Restraint stress induced changes in rat liver and serum metallothionein and in zinc metabolism, Experientia (Basel), 42 (1986a) 1006-1010. 7 J. Hidalgo, A. Armario, R. F|os, A. Dingman and J.S. Garvey, The influence of restraint stress in rats on metallothionein production and corticosterone and glucagon secretion, Life Sci., 39 (1986b) 611-616. 8 P.J. Thornalley and M. Vag~k, Possible role for mctallothionein in protection against radiation induced oxidative stress. Kinetics and mechanism of its reaction with superoxid¢ and hydroxyl radicals, Biochim. Biophys. Acta, 827 (1985) 36-44. 9 J.P. Thomas, G.J. Bachowski and A.W. Girotti, Inhibition of cell membrane lipid pcroxidation by cadmium- and zinc-metallothioneins, Biochim. Biophys. Acta, 884 (1986) 448-461. l0 J. Hidalgo, L. Campmany, M. Borr~s, J.S. Garvcy and A. Armario, Metallothioncin response to stress in rats: role in free radical scavening, Am. J. Physiol., 255 (1988a) E518-E524. I 1 K.G. Danielson, S. Ohi and P.C. Huang, Immunochemical detection of metallothionein in specific epitelial cells of rat organs, Proc. Natl. Acad. Sci., USA 79 (1982) 2301-2304. 12 I. Brcmner, J.N. Morrison, A.M. Wood and J.R. Arthur, Effects of changes in dietary zinc, copper and selenium supply and of endotoxin administration on metallothioncin I concentrations in blood cells and urine in the rat, J. Nutr., ll7 (1987) 1595-1602. 13 J.N. Morrison and I. Bremncr, Effect of maternal zinc supply on blood and tisucs mctallothioncin I concentrations in suckling rats, J. Nutr., ll7 (1987) 1588-1594. 14 M. Sato, R.K. Mehra and I. Brcmner, Measurement of plasma metallothioncin-I in assessment of zinc status of zinc-deficient and stressed rat, J. Nutr., ll4, (1984) 1683-1689. 15 J. Hidalgo, M. Giralt, J.S. Garvcy and A. Armario, Physiological role of glucocorticoids on rat serum and liver metallothionein in basal and stress conditions, Am. J. Physiol., 254 (1988b) E71-E78. 16 J.S Garvey, Metallothionein: structure/antigenicity and detection/quantitation in normal physiological fluids, Environ. Health Perspect., 54 (1984) 117-127. 17 I. Bremner and N.T. Davies, The induction of metallothionein in rat liver by zinc injection and restriction of food intake, Biochem. J., 149 (1975) 733-738. 18 A. Armario, A. Lopez-Calder6n, T. Jolin and J. Balasch, Response of anterior pituitary hormones to chronic stress. The specificity of adaptation, Neurosci. Biobehav. Rev., 10 (1986) 245-250. 19 E.A. Stone and R. McCarty, Adaptation to stress: tyrosine hydroxylase activity and catecholamines release, Neurosci. Biobehav. Rev., 7 (1983) 29-34. 20 J. Hidalgo, J.S. Garvey and A. Armario, The role of catecholamines and glucagon on serum and liver metallothionein response to restraint stress, Rev. Esp. Fisiol., 43 (1987) 433-438.