9 1985 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/85/0811-0231502.00
The Effect of Anesthetic Agents on Zinc Metabolism in the Rat Liver A. M. VAN Rid~'t AND M. T. HALL IDepartment of Surgery, East Carolina University School of Medicine, Greenville, North Carolina 27834 and 20tago University, School of Medicine, Dunedin, New Zealand
Received January 27, 1985; Accepted May 14, 1985
ABSTRACT The administration of nembutal and chloral hydrate anesthetic agents in the rat produces an increase in the uptake of zinc, as Zn-65, in the liver. Associated with this is the appearance of a lowmolecular-weight Zn-binding protein in the soluble cytosol fraction. This protein is comparable to that i n d u c e d by the stress of severe exercise, by b u r n injury, and by Zn injection, and is probably Zn metallothionein. This is an example of the induction of a Zn binding protein in the liver by a drug, and confirms that anesthesia significantly effects Zn metabolism in the liver. Consequently this effect of anesthetic agents should be taken into account in the investigation of the regulation of Zn metabolism. Index Entries: Zinc, metabolism in the rat liver; metallothionein, effect of anesthetic agents on; anesthesia, effect on Zn metabolism in the rat liver; metabolism, effect of anesthetics on Zn; rat liver, effect of anesthetics on Zn metabolism in; liver, effect of anesthetic agents on Zn metabolism in rat. *Author to whom all correspondence and reprint requests should be addressed. tCurrent address: Andr4 van Rij, MD, Department of Surgery, ,School of Medicine, Otago University, Dunedin, New Zealand. Biological Trace Element Research
23 ]
VoL 8, 1985
232
Van Rij and Hall
INTRODUCTION N u m e r o u s studies of zinc metabolism in the liver have been reported, but the associated effect of anesthetic agents c o m m o n l y used has not been taken into account. It is n o w clear that a variety of stresses, including starvation, low temperatures, exercise, and injury, affect liver Zn metabolism (1-3) and result in the production of zinc-binding proteins, metallothioneins, in the liver cytosol. The induction of Znmetallothionein was initially observed in experiments in which relatively large doses of Zn were either injected or ingested, and this was associated with Zn accumulation in the liver (4,5). Increased uptakes of hepatic zinc have also been observed with endotoxin and hydrocortisone and to a lesser extent with chloroform (6). Recently, alpha-mercaptoq3-arylacrylic acids have been observed to increase the incorporation of Zn in the liver and this was associated with metallothionein induction in the cytosol fraction (7). Consequently, c o m m o n l y used agents (such as anesthetics) in the experimental study of stress on zinc metabolism m a y modify the responses. These effects on Zn metabolism have not as yet been reported with other organic c o m p o u n d s or drugs, although drugs that interfere with liver protein synthesis do inhibit Zn accumulation and metallothionein synthesis (8,9). This study examines the effect of nembutal and chloral hydrate on Zn uptake in the liver and the associated changes in Zn binding in the hepatic cytosol fraction. METHOD Three groups of male Holtzman rats, 250-300 g, were individually h o u s e d in stainless-steel cages in a temperature- and humidity-controlled animal room. Each animal received 12 ~Ci of carrier-free Zn-65 (New England Nuclear) intraperitoneally prior to receiving either pentobarbitone sodium (50 mg/kg), chloral hydrate (300 mg/kg), or saline placebo by intraperitoneal injection. The animals were not starved prior to anesthesia and free access to food was maintained thereafter. The animals were sacrificed 2.4 h after receiving the anesthetic injection and the livers were excised. Uniform portions were taken for scintillation counting of whole tissue. For further separation of the soluble cytosol fractions, 3 g of tissue was h o m o g e n i z e d in 10 mL of 0.05M TrisHC1 and 0.25M sucrose at.pH 8.2 and was centrifuged for t .h at 100,O00g. Gel filtration of the supernatant was carried out on Sephadex G-75, 1.75 x 45 cm eluting with 0.05M Tris-HC1 at pH 8.2 and flow 1 mL/min. Statistical comparison was m a d e by use of the Student's t-test for data of normal distribution. For comparison, similar studies were carried out in animals that were either exercised to exhaustion, or given a standard 20% full thick-" Biological Trace Element Research
Vol. 8, ] 985
Anesthetic Agents and Zing Metabolism
233
ness burn, as previously described (10,11) or given 2 mg of stable zinc. Zn-65 was injected immediately prior to these events and the animals were sacrificed 24 h thereafter with the purpose of identifying the lowmolecular-weight Zn-binding protein in the cytosol.
RESULTS The uptake of Zn-65 in the liver at 24 h following anesthesia induction was significantly increased with both agents compared to the placebo controls (Table 1, p < 0.01). This increase occurred predominantly in the cell cytosol, but also to a lesser extent in the nuclear, mitochondrial, and microsomal fractions, which w e r e also separated. Following anesthesia with either agent, a low molecular weight protein (approximately 13,000 daltons) with no absorbance at 280 n m appeared in the cytosol. (b)
(a)
600
- -
400
--
/
2oo I= 1=:
I
I
0
v
@
(d)
(c) 1000 8 i
800 riO0
400 200 0 20
~ 30
I 40
E 50
60
20
30
40
50
6O
elution volume (ml)
Fig. 1. Profiles of Zn-65 in rat liver cytosol fractions separated on Sephadex g-75: (a) normal; (b) anesthetic; (c) 20% burn; (d) 2 mg zinc dose. Biological Trace Element Research
VoL 8, I985
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234
This Zn-binding protein fraction was similar to that induced by Zn injection, burn stress, and severe exercise. However, the relative proportion of the Zn-65 binding in the cytosol fractions varied with the stimulus used (Fig. 1). These parenteral anesthetic agents induced the production of zinc-metallothionein and this was associated with increased Zn-65 uptake.
DISCUSSION 'This study demonstrates yet another means by which Zn uptake into the liver may be increased with the associated induction of a Znbinding protein in the soluble cytosol cell fraction. The induction of protein synthesis by anesthetic agents is well recognized and this n o w also includes the induction of this metal binding protein. A l t h o u g h not directly characterized, this protein was observed to have a lower molecular weight with no absorption at 280 nm and was comparable to that ind u c e d by Zn injection. Consequently, it is assumed that this protein is Zn-metallothionein. Another important feature of this protein is the high cysteine content, occupying approximately 30% of the amino acid residues (5,9). Earlier observations of the induction of Zn-metallothionein by the injection of metals suggested the role of this protein to be to detoxify metal accumulated in the liver (12,13). However, the induction by more physiological events, such as starvation and thermal stress, as well as stresses of injury, exercise, or infection (1-3) have since suggested a more important physiological role for Zn-metallothionein in the regulation of Zn metabolism or the temporary storage of zinc (11,14). The significance of the appearance of this protein both in response to Zn loading and to exposure to these anesthetics is not clear. The high cysteine content of metallothionein accounts for its affinity for the inorganic Zn, but this may also be suited for the binding of some organic c o m p o u n d s that may otherwise have deleterious cellular effects. Interestingly, Zn supplements that are the most effective stimuli to metallothionein induction do have a protective, antitoxic effect on the liver w h e n exposed to toxic agents such as carbon tetrachloride (15). W h e t h e r these are related effects is not known. TABLE 1 Zn-65 Uptake in the Liver Following Pentobarbitone Sodium and Chloral Hydrate Anesthesia' Control Zn-65 cpm x 103/g
21.9 • 1.0
Penobarbitone sodium Chloral hydrate 36.0 • 4.9b
40.5 • 4.5"
"_+SE; n = 8. ~p < 0.01. Biological Trace Element Research
VoL 8, 1985
Anesthetic Agents and Zing ~4etabolism
235
The m a g n i t u d e of the r e s p o n s e b y the liver cells to i n d u c e the synthesis of Z n - b i n d i n g p r o t e i n in the cytosol does v a r y with the s t i m u l u s u s e d (Fig. 1). T h e r e are n u m e r o u s stimuli that have b e e n r e p o r t e d to h a v e this effect a n d this n o w includes anesthetic agents. C o n s e q u e n t l y , anesthetic a g e n t s m a y significantly m o d i f y the metabolism of Z n in the liver a n d p r o b a b l y also in o t h e r tissues. This is of i m p o r t a n c e in those studies of zinc m e t a b o l i s m a n d its regulation in w h i c h a n e s t h e s i a has b e e n used. The investigation of the effects of m o r e severe stresses s u c h as t r a u m a requires the use of a n e s t h e s i a a n d in the future this p r e v i o u s l y u n r e c o g n i z e d effect of s o m e a n e s t h e t i c a g e n t s on zinc metabolism s h o u l d also be taken into account.
REFERENCES 1. S. H. Oh, J. T. Deagen, P. D. Whanger, and P. G. Weswig, Am. J. Physiol. 234, E282 (1978). 2. M. C. Powanda, Y. Villarreal, E. Rodrigues, Jr., G. Oraxton, and C. R. Kennedy, Proc. Soc. Exp. Biol. Med. 163, 296 (1980). 3. A. M. van Rij, M. T. Hall, J. T. Bray, and W. J. Pories, Surg. Forum 31, 85 (1980). 4. R. W. Chen, D. J. Eakin, and P. D. Whanger, Nutr. Report Int. 10, 195 (1974). 5. I. Bremner and N. T. Davies, Biochem. J. 149, 733 (1975). 6. E. Giroux, B. DaGue and N. J. Prakash, Bioinorganic Chem. 9, 205 (1978). 7. M. P. Richards and R. J. Cousins, Proc. Soc. Exp. Biol. Med. 4, 215 (1975). 8. M. P. Richards and R. J. Cousins, Proc. Soc. Exp. Biol. Med. 156, 505 (1977). 9. H. Ohtake and M. Koga, Biochem. J. 183, 683 (1979). 10. G. L. Dohm, R. T. Williams, G. J. Kasperek, and A. M. van Rij, J. Appl. Physiol. 52, 27 (1982). 11. A. M. van Rij, M. T. Hall, J. T. Bray, and W. J. Pories, Surg. Gynecot. Obst. 153, 677 (1981). 12. P. Pulido, J. H. R. Kagi, and B. L. Vallee, Biochem. J. 5, 1768 (1966). 13. R. W. Chen, E. J. Vasey, and P. D. Whanger, f. Nutr. 107, 805 (1977). 14. S. L. Feldman and R. J. Cousins, Biochem. J. 160, 581 (1976). 15. S. Z. Cagen and C. D. Klaassen, ToxicoI. Appl. Pharm. 51, 107 (1979).
Biological Trace Element Research
VoL 8, 1985
The Effect of Anesthetic Agents on Zinc Metabolism in the Rat Liver A. M. VAN Rid~'t AND M. T. HALL IDepartment of Surgery, East Carolina University School of Medicine, Greenville, North Carolina 27834 and 20tago University, School of Medicine, Dunedin, New Zealand
Received January 27, 1985; Accepted May 14, 1985
ABSTRACT The administration of nembutal and chloral hydrate anesthetic agents in the rat produces an increase in the uptake of zinc, as Zn-65, in the liver. Associated with this is the appearance of a lowmolecular-weight Zn-binding protein in the soluble cytosol fraction. This protein is comparable to that i n d u c e d by the stress of severe exercise, by b u r n injury, and by Zn injection, and is probably Zn metallothionein. This is an example of the induction of a Zn binding protein in the liver by a drug, and confirms that anesthesia significantly effects Zn metabolism in the liver. Consequently this effect of anesthetic agents should be taken into account in the investigation of the regulation of Zn metabolism. Index Entries: Zinc, metabolism in the rat liver; metallothionein, effect of anesthetic agents on; anesthesia, effect on Zn metabolism in the rat liver; metabolism, effect of anesthetics on Zn; rat liver, effect of anesthetics on Zn metabolism in; liver, effect of anesthetic agents on Zn metabolism in rat. *Author to whom all correspondence and reprint requests should be addressed. tCurrent address: Andr4 van Rij, MD, Department of Surgery, ,School of Medicine, Otago University, Dunedin, New Zealand. Biological Trace Element Research
23 ]
VoL 8, 1985
232
Van Rij and Hall
INTRODUCTION N u m e r o u s studies of zinc metabolism in the liver have been reported, but the associated effect of anesthetic agents c o m m o n l y used has not been taken into account. It is n o w clear that a variety of stresses, including starvation, low temperatures, exercise, and injury, affect liver Zn metabolism (1-3) and result in the production of zinc-binding proteins, metallothioneins, in the liver cytosol. The induction of Znmetallothionein was initially observed in experiments in which relatively large doses of Zn were either injected or ingested, and this was associated with Zn accumulation in the liver (4,5). Increased uptakes of hepatic zinc have also been observed with endotoxin and hydrocortisone and to a lesser extent with chloroform (6). Recently, alpha-mercaptoq3-arylacrylic acids have been observed to increase the incorporation of Zn in the liver and this was associated with metallothionein induction in the cytosol fraction (7). Consequently, c o m m o n l y used agents (such as anesthetics) in the experimental study of stress on zinc metabolism m a y modify the responses. These effects on Zn metabolism have not as yet been reported with other organic c o m p o u n d s or drugs, although drugs that interfere with liver protein synthesis do inhibit Zn accumulation and metallothionein synthesis (8,9). This study examines the effect of nembutal and chloral hydrate on Zn uptake in the liver and the associated changes in Zn binding in the hepatic cytosol fraction. METHOD Three groups of male Holtzman rats, 250-300 g, were individually h o u s e d in stainless-steel cages in a temperature- and humidity-controlled animal room. Each animal received 12 ~Ci of carrier-free Zn-65 (New England Nuclear) intraperitoneally prior to receiving either pentobarbitone sodium (50 mg/kg), chloral hydrate (300 mg/kg), or saline placebo by intraperitoneal injection. The animals were not starved prior to anesthesia and free access to food was maintained thereafter. The animals were sacrificed 2.4 h after receiving the anesthetic injection and the livers were excised. Uniform portions were taken for scintillation counting of whole tissue. For further separation of the soluble cytosol fractions, 3 g of tissue was h o m o g e n i z e d in 10 mL of 0.05M TrisHC1 and 0.25M sucrose at.pH 8.2 and was centrifuged for t .h at 100,O00g. Gel filtration of the supernatant was carried out on Sephadex G-75, 1.75 x 45 cm eluting with 0.05M Tris-HC1 at pH 8.2 and flow 1 mL/min. Statistical comparison was m a d e by use of the Student's t-test for data of normal distribution. For comparison, similar studies were carried out in animals that were either exercised to exhaustion, or given a standard 20% full thick-" Biological Trace Element Research
Vol. 8, ] 985
Anesthetic Agents and Zing Metabolism
233
ness burn, as previously described (10,11) or given 2 mg of stable zinc. Zn-65 was injected immediately prior to these events and the animals were sacrificed 24 h thereafter with the purpose of identifying the lowmolecular-weight Zn-binding protein in the cytosol.
RESULTS The uptake of Zn-65 in the liver at 24 h following anesthesia induction was significantly increased with both agents compared to the placebo controls (Table 1, p < 0.01). This increase occurred predominantly in the cell cytosol, but also to a lesser extent in the nuclear, mitochondrial, and microsomal fractions, which w e r e also separated. Following anesthesia with either agent, a low molecular weight protein (approximately 13,000 daltons) with no absorbance at 280 n m appeared in the cytosol. (b)
(a)
600
- -
400
--
/
2oo I= 1=:
I
I
0
v
@
(d)
(c) 1000 8 i
800 riO0
400 200 0 20
~ 30
I 40
E 50
60
20
30
40
50
6O
elution volume (ml)
Fig. 1. Profiles of Zn-65 in rat liver cytosol fractions separated on Sephadex g-75: (a) normal; (b) anesthetic; (c) 20% burn; (d) 2 mg zinc dose. Biological Trace Element Research
VoL 8, I985
Van Rij and Haft
234
This Zn-binding protein fraction was similar to that induced by Zn injection, burn stress, and severe exercise. However, the relative proportion of the Zn-65 binding in the cytosol fractions varied with the stimulus used (Fig. 1). These parenteral anesthetic agents induced the production of zinc-metallothionein and this was associated with increased Zn-65 uptake.
DISCUSSION 'This study demonstrates yet another means by which Zn uptake into the liver may be increased with the associated induction of a Znbinding protein in the soluble cytosol cell fraction. The induction of protein synthesis by anesthetic agents is well recognized and this n o w also includes the induction of this metal binding protein. A l t h o u g h not directly characterized, this protein was observed to have a lower molecular weight with no absorption at 280 nm and was comparable to that ind u c e d by Zn injection. Consequently, it is assumed that this protein is Zn-metallothionein. Another important feature of this protein is the high cysteine content, occupying approximately 30% of the amino acid residues (5,9). Earlier observations of the induction of Zn-metallothionein by the injection of metals suggested the role of this protein to be to detoxify metal accumulated in the liver (12,13). However, the induction by more physiological events, such as starvation and thermal stress, as well as stresses of injury, exercise, or infection (1-3) have since suggested a more important physiological role for Zn-metallothionein in the regulation of Zn metabolism or the temporary storage of zinc (11,14). The significance of the appearance of this protein both in response to Zn loading and to exposure to these anesthetics is not clear. The high cysteine content of metallothionein accounts for its affinity for the inorganic Zn, but this may also be suited for the binding of some organic c o m p o u n d s that may otherwise have deleterious cellular effects. Interestingly, Zn supplements that are the most effective stimuli to metallothionein induction do have a protective, antitoxic effect on the liver w h e n exposed to toxic agents such as carbon tetrachloride (15). W h e t h e r these are related effects is not known. TABLE 1 Zn-65 Uptake in the Liver Following Pentobarbitone Sodium and Chloral Hydrate Anesthesia' Control Zn-65 cpm x 103/g
21.9 • 1.0
Penobarbitone sodium Chloral hydrate 36.0 • 4.9b
40.5 • 4.5"
"_+SE; n = 8. ~p < 0.01. Biological Trace Element Research
VoL 8, 1985
Anesthetic Agents and Zing ~4etabolism
235
The m a g n i t u d e of the r e s p o n s e b y the liver cells to i n d u c e the synthesis of Z n - b i n d i n g p r o t e i n in the cytosol does v a r y with the s t i m u l u s u s e d (Fig. 1). T h e r e are n u m e r o u s stimuli that have b e e n r e p o r t e d to h a v e this effect a n d this n o w includes anesthetic agents. C o n s e q u e n t l y , anesthetic a g e n t s m a y significantly m o d i f y the metabolism of Z n in the liver a n d p r o b a b l y also in o t h e r tissues. This is of i m p o r t a n c e in those studies of zinc m e t a b o l i s m a n d its regulation in w h i c h a n e s t h e s i a has b e e n used. The investigation of the effects of m o r e severe stresses s u c h as t r a u m a requires the use of a n e s t h e s i a a n d in the future this p r e v i o u s l y u n r e c o g n i z e d effect of s o m e a n e s t h e t i c a g e n t s on zinc metabolism s h o u l d also be taken into account.
REFERENCES 1. S. H. Oh, J. T. Deagen, P. D. Whanger, and P. G. Weswig, Am. J. Physiol. 234, E282 (1978). 2. M. C. Powanda, Y. Villarreal, E. Rodrigues, Jr., G. Oraxton, and C. R. Kennedy, Proc. Soc. Exp. Biol. Med. 163, 296 (1980). 3. A. M. van Rij, M. T. Hall, J. T. Bray, and W. J. Pories, Surg. Forum 31, 85 (1980). 4. R. W. Chen, D. J. Eakin, and P. D. Whanger, Nutr. Report Int. 10, 195 (1974). 5. I. Bremner and N. T. Davies, Biochem. J. 149, 733 (1975). 6. E. Giroux, B. DaGue and N. J. Prakash, Bioinorganic Chem. 9, 205 (1978). 7. M. P. Richards and R. J. Cousins, Proc. Soc. Exp. Biol. Med. 4, 215 (1975). 8. M. P. Richards and R. J. Cousins, Proc. Soc. Exp. Biol. Med. 156, 505 (1977). 9. H. Ohtake and M. Koga, Biochem. J. 183, 683 (1979). 10. G. L. Dohm, R. T. Williams, G. J. Kasperek, and A. M. van Rij, J. Appl. Physiol. 52, 27 (1982). 11. A. M. van Rij, M. T. Hall, J. T. Bray, and W. J. Pories, Surg. Gynecot. Obst. 153, 677 (1981). 12. P. Pulido, J. H. R. Kagi, and B. L. Vallee, Biochem. J. 5, 1768 (1966). 13. R. W. Chen, E. J. Vasey, and P. D. Whanger, f. Nutr. 107, 805 (1977). 14. S. L. Feldman and R. J. Cousins, Biochem. J. 160, 581 (1976). 15. S. Z. Cagen and C. D. Klaassen, ToxicoI. Appl. Pharm. 51, 107 (1979).
Biological Trace Element Research
VoL 8, 1985
