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[4] Nutritional and physiologic significance of metallothionein
[4]
SIGNIFICANCE OF METALLOTHIONEIN
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[4] N u t r i t i o n a l a n d P h y s i o l o g i c S i g n i f i c a n c e o f Metallothionein
By I A N
BREMNER
Introduction Metallothionein (MT) and metals are closely linked. The protein binds 7 - 10 g atoms of metal/mol, appears only to occur with its full complement of metal atoms, and its synthesis is induced by many metals by a regulated process involving increased gene transcription. It is therefore not surprising that the long list of functions proposed for MT includes the control of metal metabolism. However, there is some uncertainty as to its precise role in the handling of metals, as there have been suggestions that it is involved in such diverse processes as the control of their absorption, tissue uptake, transport, storage, and detoxification. Synthesis of MT can also be induced by many physiological and nutritional factors, including starvation and imposition of various types of physical or inflammatory stress. This has implied that the protein could have other physiological roles, such as in the acute phase response, the scavenging of free radicals, the regulation of cell differentiation, and the storage of sulfur. However, as the list of proposed functions of MT grows, it becomes increasingly difficult to believe that any one protein, even one with such unique properties, could be so versatile. It seems more likely therefore that MT has some relatively basic functions, consistent with its highly conserved structure, the existence of an MT "housekeeping" gene, and the ease with which its synthesis can be induced by a plethora of metals, hormones, and related factors.
Nutritional Factors Affecting Metallothionein Production
Metals Although MT binds to and its synthesis is induced by many metals, copper and zinc are the only ones of nutritional importance. The others, including cadmium and mercury, are nonessential metals the cytotoxic effects of which appear to be reduced by binding to MT. Such detoxification of heavy metals represents an important role for MT, although it could be an adventitious consequence of the ability of these metals to induce and bind to a protein that is primarily concerned with the metabolism of zinc and copper. METHODS IN ENZYMOLOGY, VOL. 205
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form r'-~erved.
26
INTRODUCTION
[4]
Metallothionein has been isolated as a major zinc- and copper-binding protein from many tissues, such as liver, kidneys, intestine, and pancreas. Indeed, as immunological techniques for the detection and measurement of MT have improved, it has been found in most tissues, including thymus, bone marrow, brain, and reproductive organs. It is located mainly in the cell cytosol but can also occur in the nucleus in amounts that depend on the tissue metal concentration and the stage of development. For example, the intranuclear localization of MT is particularly evident in the hepatocytes of fetal and neonatal rats but decreases with age, so that at weaning the protein is localized mainly in the cytosol. 2 Appreciable amounts of copper-MT also occur in the lysosomes and other particulate fractions of copper-loaded liver from Bedlington terriers3 and pigs. 4 In the latter case, over 80% of the hepatic copper may be present as MT, distributed evenly between the cytosol and particulate fractions. Close relationships between tissue MT and metal content have been demonstrated in humans and in domestic animals receiving normal diets. Thus liver MT and zinc concentrations are closely related in human, sheep, and calf liver, whereas in pigs of normal zinc status the best correlation is between liver MT and copper concentrations: However, it has been necessary in most experiments with rats to inject the copper or zinc or feed diets that are severely deficient in or contain excessive amounts of the metals before such correlations are seen. For example, in rats given severely zinc-deficient diets, liver and intestinal MT concentrations are rapidly reduced to nondetectable levels, whereas injection of zinc or feeding diets with very high zinc contents greatly increases tissue MT levels such that most of the additional tissue zinc is bound to MT. 6,7 Injection of copper induces liver and to a lesser extent kidney MT synthesis in rats 8 whereas feeding high-copper diets increases MT levels in kidneys but does not greatly affect those in the liver. 9 Such experiments have provided valuable insight into the factors that control MT production but have not necessarily helped in the elucidation of the nutritional and physiological significance of the protein. It is only in recent years that serious attempts have been made to I j. H. R. K~gi and Y. Kojima, Experientia, Suppl. 52, 1 (1987). 2 M. Panemangalore, D. Banerjee, S. Onosaka, and M. G. Cherian, Dev. Biol. 97, 95 (1983), 3 G. F. Johnson, A. G. Morell, R. J. Stockert, and I. Sternlieb, Hepatology 1, 243 (1981). 4 R. K. Mehra and I. Bremner, Biochem. J. 219, 539 (1984). 5 I. Bremner, Experientia, Suppl. 52, 81 (1987). 6 p. Menard, C. C. McCormick, and R. J. Cousins, J. Nutr. 111, 1358 (1981). 7 M. P. Richards and R. J. Cousins, J. Nutr. 106, 1591 (1976). 8 I. Bremner, W. G. Hoekstra, N. T. Davies, and B. W. Young, Biochem. J. 174, 883 (1978). 9 I. Bremner, R. K. Mehra, J. N. Morrison, and A. M. Wood, Biochem. J. 235, 735 (1986).
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SIGNIFICANCE OF METALLOTHIONEIN
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establish the effects of moderate and nutritionally relevant changes in dietary zinc and copper content on tissue MT concentrations. For example, liver, kidney, and pancreas MT-1 concentrations decrease within a few days of rats being given a diet containing only 3 or 6 mg zinc/kg instead of the requirement level of 12 mg/kg.10 Similarly, kidney MT levels decrease in line with the decrease in kidney copper concentrations in rats made copper deficient by feeding iron-supplemented diets, 11 but dietary copper deficiency does not affect liver MT concentrations. I° In other studies in which rats were given diets with 5, 30, or 180 mg zinc/kg and 1, 6, or 36 mg copper/kg, kidney concentrations of MT and of MT mRNA were proportional to the dietary zinc intake.12 However, hepatic concentrations of MT and MT mRNA were not greatly affected by the dietary copper and zinc content, and MT mRNA levels in the intestine increased only at the highest zinc and lowest copper intakes. It was noted that this tissue-specific response in MT production occurred primarily in the organs of absorption and excretion since this could imply that MT plays a role in the control of these processes. It seems that a primary determinant of whether a change in dietary intake of zinc affects tissue MT synthesis is the tissue zinc concentration. Only when this increases above a critical basal level does interaction occur with the promoter region in the MT gene with increased transcription of MT mRNA. The influence of changes in dietary zinc intake on tissue MT content is also evident in the maternal-fetal complex. Hepatic MT levels are often greatly elevated in fetal and neonatal animals, with the nature of the bound metal and the gestational age at which maximum concentrations are found depending on species. 5 In hamsters and rats, for example, the main metals associated with MT in fetal liver are copper and zinc, respectively. 13When maternal rats are given diets of low zinc content ,during pregnancy and lactation, liver MT and MT mRNA concentrations in the pups are reduced in line with the reduced tissue zinc contents. 14-16 In contrast, maternal copper or iron deficiency does not affect hepatic MT levels in the pups, emphasizing the fact that copper is often a less potent inducer of hepatic MT synthesis than is zinc. Although iron can bind to MT in vitro it does not do so in vivo, and changes in iron status do not have a major effect on MT production. J0 I. Bremner, J. N. Morrison, A. M. Wood, and J. R. Arthur, J. Nutr. 117, 1595 (1987). lJ R. K. Mehra and I. Bremner, Biochem. J. 213, 459 (1983). J2 T. L. Blalock, M. A. Dunn, and R. J. Cousins, J. Nutr. 118, 222 (1988). 13 A. Bakka and M. Webb, Biochem. Pharmacol. 30, 721 (1981). i4 K. R. Gallant and M. G. Cherian, Biochem. Cell Biol. 64, 8 (1986). ~5K. R. Gallant and M. G. Cherian, J. Nutr. 117, 709 (1987). 16j. N. Morrison and 1. Bremner, J. Nutr. 117, 1588 (1987).
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INTRODUCTION
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Increased hepatic synthesis of zinc MT can occur at very high dietary iron intakes although this is probably a stress response.17 Kidney MT levels may decrease in iron-loaded 11 and iron-deficient rats, 18 but this results from induced copper deficiency and anorexia, respectively. Iron deficiency does not consistently affect MT levels in the liver but increases those in the red blood cells because of the increased production of MT-rich reticulocytes.18 Other Nutrients No systematic study has been made of the effects of other changes in nutritional status on MT production. Reduction of food intake increases liver MT concentrations, probably because of the influence of glucagon and other "stress factors" on MT synthesis2 Protein deficiency also increases liver MT concentrations, even though liver zinc concentrations are decreased, but its effects on kidney MT concentrations are variable and depend on the degree of protein deprivationY Because of the high cysteine content of MT there has been some interest in the effects of dietary sulfur supply on MT production. Surprisingly, liver MTs were increased in rats given sulfydryl-deficient diets, probably because of the reduction in their food intake, indicating that sulfur is not a limiting factor for MT synthesis.2° Metallothionein in the Control of Metal Absorption The presence of MT in most cell types suggests that it plays a general role in the handling of metals. Nevertheless there have been claims that it plays a specific role in some tissues, such as in the control of metal absorption in the gastrointestinal tract. This suggestion was based on the existence of an inverse relationship between the efficiency of zinc absorption and intestinal MT concentrations. 6,7,~1 In zinc-deficient animals, which absorb zinc with high efficiency, little MT is present in the intestinal mucosa to limit zinc transfer to the plasma. Conversely, in zinc-loaded animals, where homeostatic control of zinc metabolism results in reduced zinc absorption, zinc is apparently incorporated into intestinal MT with concomitant reduction in transfer of zinc into the plasma. This attractive J7 C. C. McCormick, Proc. Soc. Exp. Biol. Med. 176, 392 (1984). ~8A. Robertson, J. N. Morrison, A. M. Wood, and I. Bremner, J. Nutr. 119, 439 (1988). ~9I. Bremner, in "Essential and Toxic Trace Elements in Human Health and Disease" (A. S. Prasad, ed.), in press. Wiley, New York, 1990. 20 L. E. Sendelbach, C. A. White, S. Howell, Z. Gregusa, and C. D. Klaassen, Toxicol. Appl. Pharmacol. 102, 259 (1990). 2~ R. J. Cousins, Physiol. Rev. 65, 238 (1985).
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hypothesis was originally based on experiments that involved parenteral injection of rats with zinc and are therefore not necessarily of nutritional relevance. Other criticisms of this hypothesis include the failure to take cognizance of specific activity changes in the endogenous mucosal zinc pool that would affect estimates of zinc absorption. 22 Moreover, no clear inverse relationship was found between mucosal MT concentrations and the efficiency of zinc absorption in mice. 22'23 Indeed, it has even been claimed that MT facilitates rather than inhibits zinc absorption. However, studies on intestinally perfused rats indicate that the mucosal buffer role of MT and the opposing view of its facilitating role in zinc absorption are not necessarily mutually exclusive views of its function in the intestine. 24 Nevertheless, when dietary zinc intakes in rats are varied over a nutritionally relevant range, where changes in the efficiency of zinc absorption have been recorded, no major changes in mucosal MT concentrations have been foundY Only with very high dietary zinc intakes do MT concentrations increase, possibly because it is only then that cellular zinc concentrations increase significantly. It is possible also that MT is then involved in the control of zinc excretion, as has been implied by some immunocytochemical investigations. 26 When dietary zinc and copper intakes by rats were varied over a limited range in one investigation, significant increases in MT and MT mRNA levels were detected only in the group receiving both a high zinc (180 mg/kg diet) and low copper (l mg/kg diet) intake, indicating that M T gene expression was highest in that group.12 However, the treatments had no effect on zinc or copper absorption. The importance of MT as a regulator of zinc absorption is still therefore far from clear. Similarly, there is no evidence that changes in dietary copper supply over a physiologically relevant range have any effect on mucosal MT concentrations, indicating that MT is not part of the homeostatic control mechanism regulating copper absorptionfl 2,27 Nevertheless, reduced copper absorption has been reported in circumstances where mucosal MT concentrations are elevated, such as in brindled mice, which have a genetic abnormality that limits copper absorption. 2s This may reflect a defect in the efflux of copper from the mucosal cells and the stimulation of MTgene transcription by the copper that accumulates in the cell. Similarly the decrease in the efficiency of copper absorption in animals given high-zinc 22 B. C. Starcher, J. G. Glauber, and J. G. Madaras, J. Nutr. 110, 1391 (1980). 23 p. R. Flanagan, J. Haist, and L. S. Valberg, J. Nutr. 113, 962 (1983). 24 j. E. Hoadley, A. S. Leinart, and R. J. Cousins, J. Nutr. 118, 497 (1988). 25 A. C. Hall, B. W. Young, and I. Bremner, J. Inorg. Biochem. 11, 57 (1979). 26 H. Nishimura, N. Nishimura, and C. Tohyama, J. Histochem. Cytochem. 37, 715 (1989). 27 p. Oestreicher and R. J. Cousins, J. Nutr. 115, 159 (1985). 28 I. J. Crane and D. M. Hunt, Chem.-Biol. Interact. 45, 113 (1983).
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diets has been attributed to the induction, by the excess zinc, of mucosal MT synthesis, with preferential binding of copper to the protein and inhibition of its transfer into the plasmaY This therefore provides a plausible explanation at a molecular level for the zinc-copper interaction. However, it has yet to be proved that copper associated with mucosal MT is unavailable to the animal and is excreted on desquamation of the intestinal cells, as is commonly assumed. Moreover, the effects of zinc on copper absorption and mucosal copper-MT accumulation have only been demonstrated in the rat at very high zinc intakes and have not been substantiated at more physiological levels. 27 Nevertheless, the protective effect of zinc against Wilson's disease is commonly attributed to induction of MT synthesis in the intestinal mucosa.
Function of Metallothionein in Control of Metal Metabolism The finding that MT production is stimulated when tissue zinc and copper concentrations are increased has indicated that it functions in the cellular detoxification or storage of the metals, especially as cytotoxic effects are more commonly encountered when the metals are bound in other forms. If MT does have a storage function it is relatively transient insofar as the protein has a short half-life and the metals are rapidly released when exposure to the metals is reduced. The elevated concentrations of MT in the liver of fetal and neonatal animals have also been regarded as evidence that MT acts as a storage reserve for copper and zinc in later life, although they could also reflect the immaturity of biliary excretory mechanisms for copper in neonates. Unfortunately little is known of the fate of metals after binding to MT. It has been reported that direct transfer of zinc and copper can occur from MT to apoenzymes such as alkaline phosphatase and superoxide dismutase, implying that MT might be involved in regulation of the activation of these and other enzymes. 29-3~ However, such transfer has only been demonstrated in vitro and there is as yet no evidence that it occurs in vivo. Indeed, transfer of copper even in vitro occurs only under oxidizing conditions. Nevertheless, the kinetics of zinc transfer from MT in Ehrlich cells do indicate that these are rate-limiting ligand substitution processes that do not involve degradation of the proteinJ ° Another possibility is that MT acts as a metal transport protein in the movement of metal between tissues. There is good evidence for such a role 29 B. L. Geller and D. R. Winge, Arch. Biochem. Biophys. 213, 109 (1982). 30 S. K. Krezoski, J. Villalobos, C. F. Shaw, and D. H. Petering, Biochem. J. 255, 483 (1988). 3~ A. O. Udom and F. O. Brady, Biochern. J. 187, 329 (1980).
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in the transport of cadmium from the liver to the kidneys, since cadmiumMT has been detected in the plasma of cadmium-exposed subjects and animals and parenterally administered cadmium-MT is selectively absorbed by the kidneys? 2 Similarly, elevated copper-MT concentrations occur in the plasma of copper-loaded animals, although this may partly reflect leakage of the protein because of copper-induced liver damage. 9 However, such secretion appears to make only a limited contribution to the loss of copper-MT from the liver. It seems likely that the fate of metals in MT depends on the particular needs of the animal and that the copper and zinc can be used for metabolic processes, deposited in new tissue, or excreted if not required. Such a concept is consistent with MT acting in the homeostatic regulation of copper and zinc metabolism. Autoregulation of M T genes serves to keep intracellular concentrations of potentially toxic free Zn 2+ and Cu 2+ at a low level and to bind the excess metal in a nontoxic form? 3 This metal may be released on degradation of the protein or in some cases may accumulate in lysosomes or other organelles in the form of insoluble aggregates of copper-MT. Alternatively the protein may be secreted in intact form from the cell or it may donate its metal to apoenzymes by ligand exchange reactions. In essence, therefore, MT acts as a metal buffer that establishes steady-state kinetics for intracellular Cu z+ and Zn 2+ levels. The autoregulation of M T genes involves binding of incoming metal ions to a small pool of thionein. However, if the influx of metal ions is excessive, they then interact with other proteins that bind to the M T genes and induce synthesis of additional thionein, which then binds the excess metal ions. The subsequent reduction in the concentration of free metal ions reduces the stimulus for increased thionein production, which then returns to basal levels. Physiological Factors Affecting MetaUothionein Synthesis The above hypothesis explains many aspects of the link between MT synthesis and metal load. However, it does not explain why MT synthesis in the liver and in some other tissues can be induced by restriction of food intake, bacterial infection, and by imposition of many types of physical and inflammatory stress (Table I)) Such induction is mediated by a range of factors that interact directly or indirectly with regulatory elements on the M T gene and increase gene transcription. Since imposition of stress increases circulating levels of glucocorticoids they have been proposed as 32 M. Nordberg and G. F. Nordberg, Experientia, Suppl. 52, 669 (1987). 33 D. H. Hamer, Annu. Rev. Biochern. 55, 913 (1986).
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TABLE I
PATHOPHYSIOLOGICALFACTORS THAT INDUCE MT SYNTHESIS Carbon tetrachloride Catecholamines Endotoxin Glucagon Glucocorticoids Infection Inflammation Interferon
Interleukin 1 Irradiation Oxidative challenge Physical stress Starvation Streptozotocin Tumor necrosis factor
inducing agents for MT synthesis. 34 Injection of rats with dexamethasone or its addition to cell cultures results in increased MT synthesis and glucocorticoid regulatory elements have been identified on the M T gene. However, there is some doubt as to the physiological importance of the glucocorticoids in stress-induced synthesis of MT. Thus chronic administration of adrenocorticotropic hormone (ACTH) to rats had no effect on basal MT concentrations in the liver and suppressed the increase caused by restraint stress. 35 Moreover, adrenalectomy or treatment with a glucocorticoid receptor blocker increased basal and stress-induced MT levels. It was claimed that glucocorticoids may on occasion have a permissive role in mobilizing MT from tissues to serum and that in physiological concentrations corticosterone has an inhibitory role in the maintenance of hepatic MT levels. 35 An alternative possibility is that stress-induced MT synthesis is caused by catecholamines, which have been shown to increase hepatic MT concentrations in rats. 36 Adrenergic blockade in male rats decreased liver MT induction by sham adrenalectomy and exogenous catecholamines but adrenoceptor blocks were ineffective in another study with female r a t s . 37-39 These results are not consistent with the relative paucity of fl receptors in male rat liver. Cyclic AMP may mediate the induction of MT synthesis by epinephrine and glucagon because analogs of this second messenger also increase liver MT levels. 2t,38,4° The effects of dexamethasone and cyclic 34 L. J. Hager and R. D. Palmiter, Nature (London) 291, 340 (1981). 35 j. Hidalgo, M. Giralt, J. S. Garvey, and A. Armario, Am. J. Physiol. 254, E71 (1988). 36 F. O. Brady and B. S. Helvig, Am. J. Physiol. 247, E318 (1984). 37 F. O. Brady, LifeSci. 28, 1647 (1981). 38 F. O. Brady, B. S. Helvig, A. E. Funk, and S. H. Garrett, Experientia, Suppl. 52, 555 (1987). 39 j. Hidalgo, M. Giralt, J. S. Garvey, and A. Armario, Horm. Metab. Res. 20, 530 (1988).
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AMP analogs on M T gene transcription are additive, suggesting that the two regulatory pathways are independent. Bacterial infection also increases hepatic MT synthesis while decreasing serum zinc concentrations, which are characteristic of an acute phase response. 41 These effects can be mimicked by administration of endotoxin, although the lipopolysaccharide is not a primary inducer of MT synthesis. Moreover, studies on transgenic mice carrying an MT-thymidine kinase fusion gene showed that the endotoxin-related promoter site for M T gene transcription is independent of the site used by glucocorticoids.42 Bacterial infection and other types of stress cause the release of the cytokine interleukin 1 (IL- 1) from monocytes and activated macrophages. This, like tumor necrosis factor and other cytokines, induces hepatic MT synthesis in rats. 43,44 IL-1 also increases MT levels in bone marrow and thymus. This tissue-specific regulation of M T gene expression appears to be responsible for the changes in zinc metabolism and particularly the increased zinc uptake by these same tissues. It has been suggested that this satisfies the increased demand in IL-1 treated rats for zinc for hematopoietic cell production. Another explanation for the induction of MT synthesis by injection involves the release of a macrophage-derived heat-stable protein factor, which is distinct from all other known inducers of MT, including IL-1.45 This factor, which is produced by macrophages in response to endotoxin exposure, stimulates MT synthesis and zinc accumulation by Chang cells in culture. 46 Although endotoxin itself does not induce MT synthesis in the human B cell line RPM1 1788, the products obtained when peripheral mononuclear cells and spleen cells are stimulated by endotoxin or concanavalin do induce the protein. 47,48 Glucagon is another possible mediator 40 M. A. Dunn and R. J. Cousins, Am. J. PhysioL 256, E420 (1989). 4t p. Z. Sobocinski, W. J. Canterbury, C. A. Mapes, and R. E. Dinterman, Am. J. Physiol. 234, E399 (1978). 42 D. M. Durnam, J. S. Hoffman, C. J. Quaife, E. P. Benditt, H. Y. Chen, and R. D. Palmiter, Proc. Natl. Acad. Sci. U.S.A. 81, 1053 (1984). 43 R. J. Cousins and A. S. Leinart, FASEB ,L 2, 2884 (1988). 44 K. L. Huber and R. J. Cousins, J. Nutr. 118, 1570 (1988). 45 y. lijima, T. Takahashi, T. Fukushima, S. Abe, Y. Itano, and F. Kosaka, Toxicol. AppL Pharmacol. 89, 135 (1987). 46 T. Fukushima, Y. Iijima, and F. Kosaka, Biochem. Biophys. Res. Commun. 152, 874 (1988). 47 S. Abe, M. Matsumi, M. Tsukioki, S. Mizukawa, T. Takahishi, I. Iijima, Y. Itano, and F. Kosaka, Experientia, Suppl. 52, 587 (1987). 48 j. Oberbarnscheidt, P. Kind, J. Abel, and E. Gleichmann, Res. Commun. Chem. Pathol. Pharmacol. 60, 211 (1988).
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INTRODUCTION
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for IL-I- or endotoxin-induced MT synthesis, since increased MT levels occur in glucagon-treated r a t s . 49 However, serum zinc levels are unaffected. The induction of MT synthesis by "stress" factors is therefore extremely complex. It may be mediated by several factors that directly or indirectly induce increased gene transcription. A common feature of the responses is increased cellular uptake and, in vivo, increased hepatic uptake of zinc. 43 In most instances this results in reduced serum zinc concentrations as part of an acute phase response. It can only be assumed that this redistribution of zinc benefits the host defense process. Metallothionein as a F r e e Radical Scavenger Another suggestion is that MT plays a role in the scavenging of free radicals, the production of which is often stimulated in "stress" conditions. Thus, treatments such as administration with IL-1 or interferon or exposure to X rays or increased oxygen tension cause increased production of oxygen free radicals and also induce MT synthesis) Production of these radicals by neutrophils and macrophages is part of the inflammatory response, designed to destroy bacteria and virus-infected cells. However, these radicals can have deleterious effects on DNA and cell membranes unless they are scavenged by some antioxidant system, such as vitamin E, glutathione, or glutathione peroxidase. Considerable interest has been shown in the possible role of MT as another free radical scavenger) ° Zinc-MT has been shown to scavenge hydroxyl radicals in vitro and to be more effective than glutathione in preventing DNA degradation by hydroxyl radicals. 51 However, zinc-MT was less effective than glutathione in inhibiting lipid peroxidation of microsomal membranes and copper-MT tended to promote free radical-mediated damage to the membranes. 52 Aerobic radiolysis of an MT solution induces metal loss and thiolate oxidation. Damage by hydroxyl radicals, which involves the metal thiolate clusters, can be reversed by lowering the pH and adding glutathione and metals. 50 The concomitant increase in lipid peroxidation and in hepatic MT levels in rats after restriction of food and water intake and their further enhancement in the presence of dimethyl sulfoxide have suggested that 49p. z. Sobocinskiand W. J. Canterbury,Ann. N.Y. Acad. Sci. 210, 354 (1982). sop. j. Thornalleyand M. Va~k, Biochim. Biophys. Acta 827, 36 (1985). 5~j. Abeland N. de Ruiter, Toxicol. Lett. 47, 191 (1989). 52J. R. Arthur,I. Bremner,P. C. Morrice,and C. F. Mills,Free Radical Res. Commun. 4, 15 (1987).
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these factors are linked. 53 However, the enhancement in MT levels caused by deprivation stress was less evident when the rats were supplemented with vitamin E, which could imply competition between MT and the vitamin in free radical scavenging. Cadmium-tolerant cells, which have increased ability to synthesize MT, tend also to have enhanced ability to resist oxidative stress? 4 Addition of zinc to hepatocyte culture also results in increased ability to withstand chemically induced free radical damage. 55 This could reflect increased cellular MT levels, although it may also be due to a stabilizing effect of zinc on membranes or effects of zinc on cytochrome P-450 or glutathione peroxidase. It has also been suggested that the cardiac toxicity of the anti-tumor drug, adriamycin, which results from extensive lipid peroxidation, can be reduced by pretreatment with metals that induce heart MT synthesis? 6 There is therefore strong circumstantial evidence that MT plays a role in the scavenging of free radicals but definitive proof has yet to be obtained. It is noteworthy that although MT synthesis is induced in different types of oxidant stress, it does not prevent the oxidative damage to tissues. Moreover, high levels of MT do not appear to suppress further MT synthesis in response to the generation of free radicals. Thus preinduction by zinc of liver MT levels does not prevent the increase in MT synthesis caused by immobilization stress. 53
53 j. Hidalgo, L. Campmany, M. Borras, J. S. Garvey, and A. Armario, Am. J. PhysioL 255, E518 (1988). 54 A. C. Mello-Filho, L. S. Chubatsu, and R. Meneghini, Biochem. J. 256, 475 (1988). 55 D. E. Coppen, D. E. Richardson, and R. J. Cousins, Proc. Soc. Exp. BioL Med. 189, 100 (1988). 56 M. Satoh, A. Naganuma, and I. Nobumasa, Toxicology 53, 231 (1988).
SIGNIFICANCE OF METALLOTHIONEIN
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[4] N u t r i t i o n a l a n d P h y s i o l o g i c S i g n i f i c a n c e o f Metallothionein
By I A N
BREMNER
Introduction Metallothionein (MT) and metals are closely linked. The protein binds 7 - 10 g atoms of metal/mol, appears only to occur with its full complement of metal atoms, and its synthesis is induced by many metals by a regulated process involving increased gene transcription. It is therefore not surprising that the long list of functions proposed for MT includes the control of metal metabolism. However, there is some uncertainty as to its precise role in the handling of metals, as there have been suggestions that it is involved in such diverse processes as the control of their absorption, tissue uptake, transport, storage, and detoxification. Synthesis of MT can also be induced by many physiological and nutritional factors, including starvation and imposition of various types of physical or inflammatory stress. This has implied that the protein could have other physiological roles, such as in the acute phase response, the scavenging of free radicals, the regulation of cell differentiation, and the storage of sulfur. However, as the list of proposed functions of MT grows, it becomes increasingly difficult to believe that any one protein, even one with such unique properties, could be so versatile. It seems more likely therefore that MT has some relatively basic functions, consistent with its highly conserved structure, the existence of an MT "housekeeping" gene, and the ease with which its synthesis can be induced by a plethora of metals, hormones, and related factors.
Nutritional Factors Affecting Metallothionein Production
Metals Although MT binds to and its synthesis is induced by many metals, copper and zinc are the only ones of nutritional importance. The others, including cadmium and mercury, are nonessential metals the cytotoxic effects of which appear to be reduced by binding to MT. Such detoxification of heavy metals represents an important role for MT, although it could be an adventitious consequence of the ability of these metals to induce and bind to a protein that is primarily concerned with the metabolism of zinc and copper. METHODS IN ENZYMOLOGY, VOL. 205
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form r'-~erved.
26
INTRODUCTION
[4]
Metallothionein has been isolated as a major zinc- and copper-binding protein from many tissues, such as liver, kidneys, intestine, and pancreas. Indeed, as immunological techniques for the detection and measurement of MT have improved, it has been found in most tissues, including thymus, bone marrow, brain, and reproductive organs. It is located mainly in the cell cytosol but can also occur in the nucleus in amounts that depend on the tissue metal concentration and the stage of development. For example, the intranuclear localization of MT is particularly evident in the hepatocytes of fetal and neonatal rats but decreases with age, so that at weaning the protein is localized mainly in the cytosol. 2 Appreciable amounts of copper-MT also occur in the lysosomes and other particulate fractions of copper-loaded liver from Bedlington terriers3 and pigs. 4 In the latter case, over 80% of the hepatic copper may be present as MT, distributed evenly between the cytosol and particulate fractions. Close relationships between tissue MT and metal content have been demonstrated in humans and in domestic animals receiving normal diets. Thus liver MT and zinc concentrations are closely related in human, sheep, and calf liver, whereas in pigs of normal zinc status the best correlation is between liver MT and copper concentrations: However, it has been necessary in most experiments with rats to inject the copper or zinc or feed diets that are severely deficient in or contain excessive amounts of the metals before such correlations are seen. For example, in rats given severely zinc-deficient diets, liver and intestinal MT concentrations are rapidly reduced to nondetectable levels, whereas injection of zinc or feeding diets with very high zinc contents greatly increases tissue MT levels such that most of the additional tissue zinc is bound to MT. 6,7 Injection of copper induces liver and to a lesser extent kidney MT synthesis in rats 8 whereas feeding high-copper diets increases MT levels in kidneys but does not greatly affect those in the liver. 9 Such experiments have provided valuable insight into the factors that control MT production but have not necessarily helped in the elucidation of the nutritional and physiological significance of the protein. It is only in recent years that serious attempts have been made to I j. H. R. K~gi and Y. Kojima, Experientia, Suppl. 52, 1 (1987). 2 M. Panemangalore, D. Banerjee, S. Onosaka, and M. G. Cherian, Dev. Biol. 97, 95 (1983), 3 G. F. Johnson, A. G. Morell, R. J. Stockert, and I. Sternlieb, Hepatology 1, 243 (1981). 4 R. K. Mehra and I. Bremner, Biochem. J. 219, 539 (1984). 5 I. Bremner, Experientia, Suppl. 52, 81 (1987). 6 p. Menard, C. C. McCormick, and R. J. Cousins, J. Nutr. 111, 1358 (1981). 7 M. P. Richards and R. J. Cousins, J. Nutr. 106, 1591 (1976). 8 I. Bremner, W. G. Hoekstra, N. T. Davies, and B. W. Young, Biochem. J. 174, 883 (1978). 9 I. Bremner, R. K. Mehra, J. N. Morrison, and A. M. Wood, Biochem. J. 235, 735 (1986).
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SIGNIFICANCE OF METALLOTHIONEIN
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establish the effects of moderate and nutritionally relevant changes in dietary zinc and copper content on tissue MT concentrations. For example, liver, kidney, and pancreas MT-1 concentrations decrease within a few days of rats being given a diet containing only 3 or 6 mg zinc/kg instead of the requirement level of 12 mg/kg.10 Similarly, kidney MT levels decrease in line with the decrease in kidney copper concentrations in rats made copper deficient by feeding iron-supplemented diets, 11 but dietary copper deficiency does not affect liver MT concentrations. I° In other studies in which rats were given diets with 5, 30, or 180 mg zinc/kg and 1, 6, or 36 mg copper/kg, kidney concentrations of MT and of MT mRNA were proportional to the dietary zinc intake.12 However, hepatic concentrations of MT and MT mRNA were not greatly affected by the dietary copper and zinc content, and MT mRNA levels in the intestine increased only at the highest zinc and lowest copper intakes. It was noted that this tissue-specific response in MT production occurred primarily in the organs of absorption and excretion since this could imply that MT plays a role in the control of these processes. It seems that a primary determinant of whether a change in dietary intake of zinc affects tissue MT synthesis is the tissue zinc concentration. Only when this increases above a critical basal level does interaction occur with the promoter region in the MT gene with increased transcription of MT mRNA. The influence of changes in dietary zinc intake on tissue MT content is also evident in the maternal-fetal complex. Hepatic MT levels are often greatly elevated in fetal and neonatal animals, with the nature of the bound metal and the gestational age at which maximum concentrations are found depending on species. 5 In hamsters and rats, for example, the main metals associated with MT in fetal liver are copper and zinc, respectively. 13When maternal rats are given diets of low zinc content ,during pregnancy and lactation, liver MT and MT mRNA concentrations in the pups are reduced in line with the reduced tissue zinc contents. 14-16 In contrast, maternal copper or iron deficiency does not affect hepatic MT levels in the pups, emphasizing the fact that copper is often a less potent inducer of hepatic MT synthesis than is zinc. Although iron can bind to MT in vitro it does not do so in vivo, and changes in iron status do not have a major effect on MT production. J0 I. Bremner, J. N. Morrison, A. M. Wood, and J. R. Arthur, J. Nutr. 117, 1595 (1987). lJ R. K. Mehra and I. Bremner, Biochem. J. 213, 459 (1983). J2 T. L. Blalock, M. A. Dunn, and R. J. Cousins, J. Nutr. 118, 222 (1988). 13 A. Bakka and M. Webb, Biochem. Pharmacol. 30, 721 (1981). i4 K. R. Gallant and M. G. Cherian, Biochem. Cell Biol. 64, 8 (1986). ~5K. R. Gallant and M. G. Cherian, J. Nutr. 117, 709 (1987). 16j. N. Morrison and 1. Bremner, J. Nutr. 117, 1588 (1987).
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Increased hepatic synthesis of zinc MT can occur at very high dietary iron intakes although this is probably a stress response.17 Kidney MT levels may decrease in iron-loaded 11 and iron-deficient rats, 18 but this results from induced copper deficiency and anorexia, respectively. Iron deficiency does not consistently affect MT levels in the liver but increases those in the red blood cells because of the increased production of MT-rich reticulocytes.18 Other Nutrients No systematic study has been made of the effects of other changes in nutritional status on MT production. Reduction of food intake increases liver MT concentrations, probably because of the influence of glucagon and other "stress factors" on MT synthesis2 Protein deficiency also increases liver MT concentrations, even though liver zinc concentrations are decreased, but its effects on kidney MT concentrations are variable and depend on the degree of protein deprivationY Because of the high cysteine content of MT there has been some interest in the effects of dietary sulfur supply on MT production. Surprisingly, liver MTs were increased in rats given sulfydryl-deficient diets, probably because of the reduction in their food intake, indicating that sulfur is not a limiting factor for MT synthesis.2° Metallothionein in the Control of Metal Absorption The presence of MT in most cell types suggests that it plays a general role in the handling of metals. Nevertheless there have been claims that it plays a specific role in some tissues, such as in the control of metal absorption in the gastrointestinal tract. This suggestion was based on the existence of an inverse relationship between the efficiency of zinc absorption and intestinal MT concentrations. 6,7,~1 In zinc-deficient animals, which absorb zinc with high efficiency, little MT is present in the intestinal mucosa to limit zinc transfer to the plasma. Conversely, in zinc-loaded animals, where homeostatic control of zinc metabolism results in reduced zinc absorption, zinc is apparently incorporated into intestinal MT with concomitant reduction in transfer of zinc into the plasma. This attractive J7 C. C. McCormick, Proc. Soc. Exp. Biol. Med. 176, 392 (1984). ~8A. Robertson, J. N. Morrison, A. M. Wood, and I. Bremner, J. Nutr. 119, 439 (1988). ~9I. Bremner, in "Essential and Toxic Trace Elements in Human Health and Disease" (A. S. Prasad, ed.), in press. Wiley, New York, 1990. 20 L. E. Sendelbach, C. A. White, S. Howell, Z. Gregusa, and C. D. Klaassen, Toxicol. Appl. Pharmacol. 102, 259 (1990). 2~ R. J. Cousins, Physiol. Rev. 65, 238 (1985).
[4]
SIGNIFICANCE OF METALLOTHIONEIN
29
hypothesis was originally based on experiments that involved parenteral injection of rats with zinc and are therefore not necessarily of nutritional relevance. Other criticisms of this hypothesis include the failure to take cognizance of specific activity changes in the endogenous mucosal zinc pool that would affect estimates of zinc absorption. 22 Moreover, no clear inverse relationship was found between mucosal MT concentrations and the efficiency of zinc absorption in mice. 22'23 Indeed, it has even been claimed that MT facilitates rather than inhibits zinc absorption. However, studies on intestinally perfused rats indicate that the mucosal buffer role of MT and the opposing view of its facilitating role in zinc absorption are not necessarily mutually exclusive views of its function in the intestine. 24 Nevertheless, when dietary zinc intakes in rats are varied over a nutritionally relevant range, where changes in the efficiency of zinc absorption have been recorded, no major changes in mucosal MT concentrations have been foundY Only with very high dietary zinc intakes do MT concentrations increase, possibly because it is only then that cellular zinc concentrations increase significantly. It is possible also that MT is then involved in the control of zinc excretion, as has been implied by some immunocytochemical investigations. 26 When dietary zinc and copper intakes by rats were varied over a limited range in one investigation, significant increases in MT and MT mRNA levels were detected only in the group receiving both a high zinc (180 mg/kg diet) and low copper (l mg/kg diet) intake, indicating that M T gene expression was highest in that group.12 However, the treatments had no effect on zinc or copper absorption. The importance of MT as a regulator of zinc absorption is still therefore far from clear. Similarly, there is no evidence that changes in dietary copper supply over a physiologically relevant range have any effect on mucosal MT concentrations, indicating that MT is not part of the homeostatic control mechanism regulating copper absorptionfl 2,27 Nevertheless, reduced copper absorption has been reported in circumstances where mucosal MT concentrations are elevated, such as in brindled mice, which have a genetic abnormality that limits copper absorption. 2s This may reflect a defect in the efflux of copper from the mucosal cells and the stimulation of MTgene transcription by the copper that accumulates in the cell. Similarly the decrease in the efficiency of copper absorption in animals given high-zinc 22 B. C. Starcher, J. G. Glauber, and J. G. Madaras, J. Nutr. 110, 1391 (1980). 23 p. R. Flanagan, J. Haist, and L. S. Valberg, J. Nutr. 113, 962 (1983). 24 j. E. Hoadley, A. S. Leinart, and R. J. Cousins, J. Nutr. 118, 497 (1988). 25 A. C. Hall, B. W. Young, and I. Bremner, J. Inorg. Biochem. 11, 57 (1979). 26 H. Nishimura, N. Nishimura, and C. Tohyama, J. Histochem. Cytochem. 37, 715 (1989). 27 p. Oestreicher and R. J. Cousins, J. Nutr. 115, 159 (1985). 28 I. J. Crane and D. M. Hunt, Chem.-Biol. Interact. 45, 113 (1983).
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diets has been attributed to the induction, by the excess zinc, of mucosal MT synthesis, with preferential binding of copper to the protein and inhibition of its transfer into the plasmaY This therefore provides a plausible explanation at a molecular level for the zinc-copper interaction. However, it has yet to be proved that copper associated with mucosal MT is unavailable to the animal and is excreted on desquamation of the intestinal cells, as is commonly assumed. Moreover, the effects of zinc on copper absorption and mucosal copper-MT accumulation have only been demonstrated in the rat at very high zinc intakes and have not been substantiated at more physiological levels. 27 Nevertheless, the protective effect of zinc against Wilson's disease is commonly attributed to induction of MT synthesis in the intestinal mucosa.
Function of Metallothionein in Control of Metal Metabolism The finding that MT production is stimulated when tissue zinc and copper concentrations are increased has indicated that it functions in the cellular detoxification or storage of the metals, especially as cytotoxic effects are more commonly encountered when the metals are bound in other forms. If MT does have a storage function it is relatively transient insofar as the protein has a short half-life and the metals are rapidly released when exposure to the metals is reduced. The elevated concentrations of MT in the liver of fetal and neonatal animals have also been regarded as evidence that MT acts as a storage reserve for copper and zinc in later life, although they could also reflect the immaturity of biliary excretory mechanisms for copper in neonates. Unfortunately little is known of the fate of metals after binding to MT. It has been reported that direct transfer of zinc and copper can occur from MT to apoenzymes such as alkaline phosphatase and superoxide dismutase, implying that MT might be involved in regulation of the activation of these and other enzymes. 29-3~ However, such transfer has only been demonstrated in vitro and there is as yet no evidence that it occurs in vivo. Indeed, transfer of copper even in vitro occurs only under oxidizing conditions. Nevertheless, the kinetics of zinc transfer from MT in Ehrlich cells do indicate that these are rate-limiting ligand substitution processes that do not involve degradation of the proteinJ ° Another possibility is that MT acts as a metal transport protein in the movement of metal between tissues. There is good evidence for such a role 29 B. L. Geller and D. R. Winge, Arch. Biochem. Biophys. 213, 109 (1982). 30 S. K. Krezoski, J. Villalobos, C. F. Shaw, and D. H. Petering, Biochem. J. 255, 483 (1988). 3~ A. O. Udom and F. O. Brady, Biochern. J. 187, 329 (1980).
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SIGNIFICANCEOF METALLOTHIONEIN
31
in the transport of cadmium from the liver to the kidneys, since cadmiumMT has been detected in the plasma of cadmium-exposed subjects and animals and parenterally administered cadmium-MT is selectively absorbed by the kidneys? 2 Similarly, elevated copper-MT concentrations occur in the plasma of copper-loaded animals, although this may partly reflect leakage of the protein because of copper-induced liver damage. 9 However, such secretion appears to make only a limited contribution to the loss of copper-MT from the liver. It seems likely that the fate of metals in MT depends on the particular needs of the animal and that the copper and zinc can be used for metabolic processes, deposited in new tissue, or excreted if not required. Such a concept is consistent with MT acting in the homeostatic regulation of copper and zinc metabolism. Autoregulation of M T genes serves to keep intracellular concentrations of potentially toxic free Zn 2+ and Cu 2+ at a low level and to bind the excess metal in a nontoxic form? 3 This metal may be released on degradation of the protein or in some cases may accumulate in lysosomes or other organelles in the form of insoluble aggregates of copper-MT. Alternatively the protein may be secreted in intact form from the cell or it may donate its metal to apoenzymes by ligand exchange reactions. In essence, therefore, MT acts as a metal buffer that establishes steady-state kinetics for intracellular Cu z+ and Zn 2+ levels. The autoregulation of M T genes involves binding of incoming metal ions to a small pool of thionein. However, if the influx of metal ions is excessive, they then interact with other proteins that bind to the M T genes and induce synthesis of additional thionein, which then binds the excess metal ions. The subsequent reduction in the concentration of free metal ions reduces the stimulus for increased thionein production, which then returns to basal levels. Physiological Factors Affecting MetaUothionein Synthesis The above hypothesis explains many aspects of the link between MT synthesis and metal load. However, it does not explain why MT synthesis in the liver and in some other tissues can be induced by restriction of food intake, bacterial infection, and by imposition of many types of physical and inflammatory stress (Table I)) Such induction is mediated by a range of factors that interact directly or indirectly with regulatory elements on the M T gene and increase gene transcription. Since imposition of stress increases circulating levels of glucocorticoids they have been proposed as 32 M. Nordberg and G. F. Nordberg, Experientia, Suppl. 52, 669 (1987). 33 D. H. Hamer, Annu. Rev. Biochern. 55, 913 (1986).
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TABLE I
PATHOPHYSIOLOGICALFACTORS THAT INDUCE MT SYNTHESIS Carbon tetrachloride Catecholamines Endotoxin Glucagon Glucocorticoids Infection Inflammation Interferon
Interleukin 1 Irradiation Oxidative challenge Physical stress Starvation Streptozotocin Tumor necrosis factor
inducing agents for MT synthesis. 34 Injection of rats with dexamethasone or its addition to cell cultures results in increased MT synthesis and glucocorticoid regulatory elements have been identified on the M T gene. However, there is some doubt as to the physiological importance of the glucocorticoids in stress-induced synthesis of MT. Thus chronic administration of adrenocorticotropic hormone (ACTH) to rats had no effect on basal MT concentrations in the liver and suppressed the increase caused by restraint stress. 35 Moreover, adrenalectomy or treatment with a glucocorticoid receptor blocker increased basal and stress-induced MT levels. It was claimed that glucocorticoids may on occasion have a permissive role in mobilizing MT from tissues to serum and that in physiological concentrations corticosterone has an inhibitory role in the maintenance of hepatic MT levels. 35 An alternative possibility is that stress-induced MT synthesis is caused by catecholamines, which have been shown to increase hepatic MT concentrations in rats. 36 Adrenergic blockade in male rats decreased liver MT induction by sham adrenalectomy and exogenous catecholamines but adrenoceptor blocks were ineffective in another study with female r a t s . 37-39 These results are not consistent with the relative paucity of fl receptors in male rat liver. Cyclic AMP may mediate the induction of MT synthesis by epinephrine and glucagon because analogs of this second messenger also increase liver MT levels. 2t,38,4° The effects of dexamethasone and cyclic 34 L. J. Hager and R. D. Palmiter, Nature (London) 291, 340 (1981). 35 j. Hidalgo, M. Giralt, J. S. Garvey, and A. Armario, Am. J. Physiol. 254, E71 (1988). 36 F. O. Brady and B. S. Helvig, Am. J. Physiol. 247, E318 (1984). 37 F. O. Brady, LifeSci. 28, 1647 (1981). 38 F. O. Brady, B. S. Helvig, A. E. Funk, and S. H. Garrett, Experientia, Suppl. 52, 555 (1987). 39 j. Hidalgo, M. Giralt, J. S. Garvey, and A. Armario, Horm. Metab. Res. 20, 530 (1988).
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33
AMP analogs on M T gene transcription are additive, suggesting that the two regulatory pathways are independent. Bacterial infection also increases hepatic MT synthesis while decreasing serum zinc concentrations, which are characteristic of an acute phase response. 41 These effects can be mimicked by administration of endotoxin, although the lipopolysaccharide is not a primary inducer of MT synthesis. Moreover, studies on transgenic mice carrying an MT-thymidine kinase fusion gene showed that the endotoxin-related promoter site for M T gene transcription is independent of the site used by glucocorticoids.42 Bacterial infection and other types of stress cause the release of the cytokine interleukin 1 (IL- 1) from monocytes and activated macrophages. This, like tumor necrosis factor and other cytokines, induces hepatic MT synthesis in rats. 43,44 IL-1 also increases MT levels in bone marrow and thymus. This tissue-specific regulation of M T gene expression appears to be responsible for the changes in zinc metabolism and particularly the increased zinc uptake by these same tissues. It has been suggested that this satisfies the increased demand in IL-1 treated rats for zinc for hematopoietic cell production. Another explanation for the induction of MT synthesis by injection involves the release of a macrophage-derived heat-stable protein factor, which is distinct from all other known inducers of MT, including IL-1.45 This factor, which is produced by macrophages in response to endotoxin exposure, stimulates MT synthesis and zinc accumulation by Chang cells in culture. 46 Although endotoxin itself does not induce MT synthesis in the human B cell line RPM1 1788, the products obtained when peripheral mononuclear cells and spleen cells are stimulated by endotoxin or concanavalin do induce the protein. 47,48 Glucagon is another possible mediator 40 M. A. Dunn and R. J. Cousins, Am. J. PhysioL 256, E420 (1989). 4t p. Z. Sobocinski, W. J. Canterbury, C. A. Mapes, and R. E. Dinterman, Am. J. Physiol. 234, E399 (1978). 42 D. M. Durnam, J. S. Hoffman, C. J. Quaife, E. P. Benditt, H. Y. Chen, and R. D. Palmiter, Proc. Natl. Acad. Sci. U.S.A. 81, 1053 (1984). 43 R. J. Cousins and A. S. Leinart, FASEB ,L 2, 2884 (1988). 44 K. L. Huber and R. J. Cousins, J. Nutr. 118, 1570 (1988). 45 y. lijima, T. Takahashi, T. Fukushima, S. Abe, Y. Itano, and F. Kosaka, Toxicol. AppL Pharmacol. 89, 135 (1987). 46 T. Fukushima, Y. Iijima, and F. Kosaka, Biochem. Biophys. Res. Commun. 152, 874 (1988). 47 S. Abe, M. Matsumi, M. Tsukioki, S. Mizukawa, T. Takahishi, I. Iijima, Y. Itano, and F. Kosaka, Experientia, Suppl. 52, 587 (1987). 48 j. Oberbarnscheidt, P. Kind, J. Abel, and E. Gleichmann, Res. Commun. Chem. Pathol. Pharmacol. 60, 211 (1988).
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INTRODUCTION
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for IL-I- or endotoxin-induced MT synthesis, since increased MT levels occur in glucagon-treated r a t s . 49 However, serum zinc levels are unaffected. The induction of MT synthesis by "stress" factors is therefore extremely complex. It may be mediated by several factors that directly or indirectly induce increased gene transcription. A common feature of the responses is increased cellular uptake and, in vivo, increased hepatic uptake of zinc. 43 In most instances this results in reduced serum zinc concentrations as part of an acute phase response. It can only be assumed that this redistribution of zinc benefits the host defense process. Metallothionein as a F r e e Radical Scavenger Another suggestion is that MT plays a role in the scavenging of free radicals, the production of which is often stimulated in "stress" conditions. Thus, treatments such as administration with IL-1 or interferon or exposure to X rays or increased oxygen tension cause increased production of oxygen free radicals and also induce MT synthesis) Production of these radicals by neutrophils and macrophages is part of the inflammatory response, designed to destroy bacteria and virus-infected cells. However, these radicals can have deleterious effects on DNA and cell membranes unless they are scavenged by some antioxidant system, such as vitamin E, glutathione, or glutathione peroxidase. Considerable interest has been shown in the possible role of MT as another free radical scavenger) ° Zinc-MT has been shown to scavenge hydroxyl radicals in vitro and to be more effective than glutathione in preventing DNA degradation by hydroxyl radicals. 51 However, zinc-MT was less effective than glutathione in inhibiting lipid peroxidation of microsomal membranes and copper-MT tended to promote free radical-mediated damage to the membranes. 52 Aerobic radiolysis of an MT solution induces metal loss and thiolate oxidation. Damage by hydroxyl radicals, which involves the metal thiolate clusters, can be reversed by lowering the pH and adding glutathione and metals. 50 The concomitant increase in lipid peroxidation and in hepatic MT levels in rats after restriction of food and water intake and their further enhancement in the presence of dimethyl sulfoxide have suggested that 49p. z. Sobocinskiand W. J. Canterbury,Ann. N.Y. Acad. Sci. 210, 354 (1982). sop. j. Thornalleyand M. Va~k, Biochim. Biophys. Acta 827, 36 (1985). 5~j. Abeland N. de Ruiter, Toxicol. Lett. 47, 191 (1989). 52J. R. Arthur,I. Bremner,P. C. Morrice,and C. F. Mills,Free Radical Res. Commun. 4, 15 (1987).
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SIGNIFICANCE OF METALLOTHIONEIN
35
these factors are linked. 53 However, the enhancement in MT levels caused by deprivation stress was less evident when the rats were supplemented with vitamin E, which could imply competition between MT and the vitamin in free radical scavenging. Cadmium-tolerant cells, which have increased ability to synthesize MT, tend also to have enhanced ability to resist oxidative stress? 4 Addition of zinc to hepatocyte culture also results in increased ability to withstand chemically induced free radical damage. 55 This could reflect increased cellular MT levels, although it may also be due to a stabilizing effect of zinc on membranes or effects of zinc on cytochrome P-450 or glutathione peroxidase. It has also been suggested that the cardiac toxicity of the anti-tumor drug, adriamycin, which results from extensive lipid peroxidation, can be reduced by pretreatment with metals that induce heart MT synthesis? 6 There is therefore strong circumstantial evidence that MT plays a role in the scavenging of free radicals but definitive proof has yet to be obtained. It is noteworthy that although MT synthesis is induced in different types of oxidant stress, it does not prevent the oxidative damage to tissues. Moreover, high levels of MT do not appear to suppress further MT synthesis in response to the generation of free radicals. Thus preinduction by zinc of liver MT levels does not prevent the increase in MT synthesis caused by immobilization stress. 53
53 j. Hidalgo, L. Campmany, M. Borras, J. S. Garvey, and A. Armario, Am. J. PhysioL 255, E518 (1988). 54 A. C. Mello-Filho, L. S. Chubatsu, and R. Meneghini, Biochem. J. 256, 475 (1988). 55 D. E. Coppen, D. E. Richardson, and R. J. Cousins, Proc. Soc. Exp. BioL Med. 189, 100 (1988). 56 M. Satoh, A. Naganuma, and I. Nobumasa, Toxicology 53, 231 (1988).