Role of Metallothioneins in Copper Transport in Patients with Menkes Syndrome

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 8, No. 4 Copyright 1978, Institute for Clinical Science Role of Metallothioneins in Copper Transport in Patients with Menkes Syndrome ADOLFO D. GARNICA, M.D., W. Y. CHAN, Ph.D.* and OW EN M. RENNERT, M.D.* Division of Genetics, Department of Pediatrics, University o f Florida College of Medicine, Gainesville, FL and Department o f Pediatrics, Health Science Center, University of Oklahoma, Oklahoma City, OK ABSTRACT Fibroblasts from infants w ith M enkes kinky hair syndrom e, which accum u late excessive quantities of copper, are thought to represent a disorder of copper storage or transport. Because of this abnormality, it was thought that they might provide a useful system for investigation of the presum ed storage or transport protein metallothionein. Data are presented which are consistent with defective copper efflux from the m utant cells. Because of the more specific role of m etallothionein in cadm ium detoxification, studies of cadmium metabolism were undertaken which dem onstrated abnormal cadmium retention and m etallothionein induction in the mutant cells. The association, therefore, of a defect of cadmium metabolism and storage with an abnormality of copper efflux provides evidence implicating m etallothionein in copper transport for fibroblasts. Introduction The m etallothioneins are zinc, copper and cadm ium-binding intracellular proteins of low molecular weight, which contain 30 to 35 percent cysteine and 6 to 11 percent m etal bound to cysteinyl side chains by m ercaptide bonds. They occur in multiple tissues in some species, in forms of varying amino acid composition, despite which their biological function has not been established.20,22 In human liver, zinc is the principal m etal constituent, although copper and cadm ium also constitute significant com ponents. M etallothionein synthesis in response to metal loading in animals implicates them in heavy metal detoxification and/or essential trace metal m etabolism.5, 10, 21, 28,30 M etallothionein synthesis occurs in response to toxic metal challenge. However, the liver content of zinc, an essential trace metal, has been reported to be an important determ inant of the concentration of metallothionein-type proteins.1,5,11,31 The function of m etallothionein in in tracellular copper transport was suggested by the identification and isolation of a /78/ $01.20 Institute for Clinical Science, Inc.

2 RO LE O F M ETA LL O TH IO N E IN S IN PA TIEN TS W IT H M ENKES SYNDROME cytosol protein of an estim ated 10,000 m olecular w eight which apparently is involved in in testin al copper absorption.9, 10,36,37 A copper-binding cytosol protein of molecular weight 11,000, the same estim ated size as m etallothionein, has been implicated in the cellular uptake of copper.36,37 On the basis of similarities betw een this protein fraction and the metallothioneins, a role has been postulated for m etallothionein in intracellular copper transport.9,10,37 Copper is potentially toxic to all forms of life, especially those having no capacity to limit its uptake. In such instances, su scep tib ility to co p p er toxicity, of necessity, becomes a function of cellular excretory efficiency. The viability of cultured fibroblasts, despite their perm eability to copper, thus implies the function of efficient homeostatic mechanisms, and the accumulation of excessive amounts of copper by skin fibroblasts from individuals with M enkes kinky hair syndrome m ight represent a failure of such m echanisms.6,13,14,16 This report reviews studies attempting to clarify the molecular basis of this abnormality and the role of metallothionein in its pathogenesis. A decrease in copper efflux from Menkes syndrome fibroblasts is recorded, along w ith an aberrant response to cadmium challenge, which implies an inborn defect in copper and cadmium m etabolism. T hese observations support the intracellular detoxification and transport functions postulated for m etallothionein.5,1u21,32,35 M aterials and Methods Skin fibroblast cultures were explanted from three infants w'ith M enkes kinky hair syndrome. Two additional m utant cultures and control cultures w ere obtained from the Human G enetic Cell Repository and the American Type Culture Collection. 64Cu was obtained from the N ew E ngland N uclear and 109C d from Amersham/Searle. 64 Cu and 109 Cd were counted in a gamma counter.* M etal analysis was performed with an atomic absorption spectrophotom eterf e q u ip ped with graphite furnace. Protein was d eterm in ed by the m ethod of Low ry et a l.23 C o p p e r -D o s e R e s p o n s e Copper sulfate was added to confluent fibroblast cultures to achieve copper concentrations of a 2 to 40 ju.g per ml. The cultures were incubated for 48 hours at 37 C, trypsinized and homogenized, and the su p e rn a ta n t copper and p ro tein measured. C a d m iu m C o n t e n t Cadmium chloride was added to basal culture m edium to achieve cadm ium concentrations of 0.1 to 1.1 meg per ml. Confluent fibroblast cultures w ere incubated for 48 hours at 37 C in medium of increasing cadmium concentration, then harvested, hom ogenized and the supernatant protein and cadm ium determ ined. C a d m iu m a n d C o p p e r U p t a k e Precisely 17 ju.ci o f64cu were added to confluent cultures in 10 ml of medium, the Cu++ concentration of which was adjusted to 9 /xg per ml with C u S 0 4. Cadmium, if present, was added to a concentration of 0.09 fig per ml. The cultures w ere incubated at 37 C for one to 10 hours, harvested, hom ogenized, the protein content of the lysate determ ined and the 64Cu counted. 109 C d uptake by cultured fibroblasts were examined at 37 C. Four nci o f109 Cd were added to the culture medium and the C d++ concentration adjusted to 0.56 ng per ml with CdCl2. * Packard Model S210. t Perkin-Elmer Model 306.

3 304 GARNICA, CHAN AND RENN ERT C o pper E f f l u x Cultures were pulse-labeled with 16.7 ju.ci of 64Cu at 37 C for 20 hours, then transferred to non-radioactive medium. Cultures were harvested and analyzed for 64 Cu and protein one to 10 hours after the m edium change. Statistical differences were examined for significance using a t-test for paired data. Results F ib r o b l a st C o p p e r a n d C adm ium C o n t e n t Cultured Menkes syndrome fibroblasts are morphologically normal but have a dim inished tolerance to copper.4 The increased sensitivity of these cells to copper toxicity is associated with increased intracellular copper content.13,16 In basal culture m edium (copper concentration ± 20.0 ng per ml) the copper content of normal cells was 108 ng per mg cell protein, while that of the m utant fibroblasts was 310 ng per mg (table I). As the copper concentration of the medium was gradually increased, the copper content of neither cell culture increased significantly until the m edium copper concentration exceeded 6 m eg per ml. At concentrations of 10 through 15 meg per ml, the copper content of both cultures increased above the baseline levels, while that of the M enkes syndrom e cells rem ained greater than the norm al (P < 0.04). At medium concentrations equal to or above 20 meg per ml, the copper conten t of the normal cells was not significantly different from that of the mutant cells. These observations suggest that copper uptake by the m utant fibroblasts is at least equal to that of normal fibroblasts. Furtherm ore, they imply that the copper regulatory m echanism s in normal fibroblasts function effectively up to a medium copper concentration of approximately 15 meg per ml, while those of Menkes syndrome fibroblasts are abnormal at all conconcentrations. An inconsistency in the general relationship betw een medium copper concentration and fibroblast copper content involving both cell lines is apparent at a medium concentration of 1 meg per ml, which most plausibly may be explained in terms of a ten-fold error in dilution, since the values at one and 10 meg per ml are essentially the same for both cultures. As the cadmium concentration of the culture m edium was increased from basal T A B L E I Copper and Cadmium Content of Cultured Skin Fibroblasts M edium (Cu++) C o p p e r C o n te n t*+ M edium (Cd++) Cadmium C o n te n t * meg p e r ml MS* (n=7) Normal (n=4) meg p e r ml MS (n - 5 ) Norm al (n=4) ± ! ± ± ± * U n i t s e x p r e s s e d a s n g m e t a l p e r mg c e l l p r o t e i n. t P < P < xm e n k e s k i n k y h a i r s y n d r o m e. l iv a l u e s e x p r e s s e d a r e m e a n s ± s t a n d a r d d e v i a t i o n.

4 ROLE O F M ETA LLOTH IONEINS IN PATIENTS W ITH MENKES SYNDROME 305 levels, so did the cadmium content (ng Cd per mg cell protein) of the normal and Menkes syndrome fibroblasts (table I). They rem ained essentially equal, how ever, up to a medium concentration of 0.3 meg per ml, when the cadmium content of the mutant cell line increased sharply. This did not occur in the normal culture until the medium concentration reached 0.5 meg p e r ml. At m edium concentrations of 0.5 to 1.1 meg per ml the fibroblast cadmium content of both cultures stabilized, with the cadmium content of the mutant fibroblasts remaining greater than that of the normal fibroblasts (P < 0.05). C o p p e r a n d C a d m iu m U p t a k e In order to investigate fu rth er the mechanism underlying the high copper content of Menkes syndrome fibroblasts, the uptake of radioactive copper and cadm ium was m easured. A relatively high medium copper concentration was used to maximize the differences in copper homeostasis betw een the two cell types, but it was m aintained sufficiently below cytotoxic levels to avoid artifactual values resulting from cell injury. During the ten-hour observation period, the 64 Cu activity of norm al skin fibroblasts did not vary significantly, possibly reflecting a stable equilibrium betw een 64Cu uptake and discharge (table II). The 64Cu activity of M enkes syndrom e fibroblasts, however, did not become stable until after seven hours at a level approximately twice normal. The 64 Cu activity of the mutant fibroblasts at one and five hours was two to three times that of the normal fibroblasts. At three hours, however, the 84Cu activity in M enkes syndrom e fibroblasts was approxim ately equal to that of the normal fibroblast. This observation might be explained in terms of a rapid influx of 64 Cu into the Menkes syndrome cells driven by a concentration gradient, which displaces unlabeled copper during the first hour. This TABLE I I Copper-64 and Cadmium-109 Incorporation by Skin Fibroblasts at 37 C Time &Vopper*t 109Cadjniuin*/íF Hrs. MS* (n=3) Normal (n=4) MSX (n=3) Normal (n=3) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.6 *10~:i x 61+Cu cpm per mg cell protein or 109Cd cpm per mg cell protein. tspecific activity &4Cu, 910 uci per mg. P < 0.05 ^Values expressed as means ± standard deviation. 1TP < 0.02 *Menkes kinky hair syndrome. is followed by a rapid shift intracellularly of the displaced copper and, finally, reequilibration and stabilization. In all but the third hour, 64Cu incorporation was consistently equal to or greater than into m utant fibroblasts than into norm al fibroblasts (P <0.05). These results substantiate the observation that copper influx into Menkes syndrome fibroblasts is not decreased and are consistent with reports of copper uptake and absorption, which indicate that the uptake of copper into intestinal mucosal cells and other tissues of patients w ith M enkes syndrom e is in tact.6' 8,14,17 Assuming that the removal of copper from cultured fibroblasts is, to some degree, a function of intracellular copper concentration, these observations suggest that the copper excretory m echanisms of M enkes syndrome fibroblasts function less effectively than those of normal cells. Incubation of normal fibroblasts with 109 Cd-containing medium demonstrated a constant, stable level of 109Cd-radioactivity throughout the study period (table II). T h e109 Cd radioactivity retained by the Menkes syndrome fibroblasts, however, was cons istently higher than in the normal fibroblasts and increased with incubation time (P < 0.02), corroborating the abnormality in transport or storage im plied by the increased cadmium content of the mutant cells (table I).

5 3 0 6 GARNICA, CHAN AND RENNERT C o p p e r R e t e n t io n To clarify whether the defect in Menkes syndrom e fibroblasts involved copper uptake or excretion,64 Cu efflux was studied by the pulse-labelling of cultured cells. During this phase of the study, skin fibroblasts were first grown in 64Cu-containing medium, which was then replaced by unlabeled m edium, the cells harvested serially after transfer and counted for 64 Cu activity. Any defect in efflux would thus appear as an increase in retained radioactivity. At 37 C, 64Cu retention was greater in the m utant than in the normal fibroblasts, except during the first and third hours after transfer to non-radioactive m edium (table III). At one and three hours, the retention of64 Cu by normal and mutant cells was essentially equal. At five hours and beyond, how ever,64 Cu retention in the Menkes syndrome fibroblasts was greater than in normal cells (P < 0.025). Since the Menkes syndrome and norm al cultures w ere exposed to nonradioactive basal m edium during the chase, any difference in 64 Cu retention may be attributed to differences in copper efflux. Thus, at 37 C, the M enkes syndrome cells dem onstrate evidence of impaired copper efflux. T A B L E III Copper-64 Retention by Cultured Fibroblasts at 37 C*+ Time o f Chase (H rs.) MS* (n=4) Normal (n=4) ±1726 / 4670 ± ±8.0 / 25.7 ± ± ± ± ± ± ± ± ± 4.0 Specific activity of cl+cu, 595 pci per mg. tp < Units expressed, ^4Cu cpm per mg cell protein, /values expressed as means ± standard deviation. U/.fter zero time, values expressed are percentages of zero time counts. xmenkes kinky hair syndrome. C o p p e r -C a d m iu m I n t e r a c t io n In vivo anim al studies have dem onstrated an interaction betw een cadmium and copper m etabolism.2,3 15,18,25,26 Simultaneous administration of cadmium and copper salts to rats results in a decrease in cadm ium sequestration by metallothionein and an increase in cadm ium toxicity.18,26 Assuming, therefore, a role for the m etallothioneins in fibroblast copper m etabolism or hom eostasis,64 Cu uptake or incorporation might be expected to be influenced by the presence o f cadm ium in the cultu re m edium. To m inim ize the effects of variation in experim ental conditions, the influence of cadm ium on copper rete n tio n is exp ressed as th e ratio o f 64Cu incorporation in M enkes sy n d ro m e : norm al fibroblasts. In the presence of 0.09 ng per nl of cadmium, copper uptake by both fibroblast lines is increased (table IV). 64 Cu uptake, however, is increased to a greater d eg ree in norm al fibroblasts than in Menkes syndrome fibroblasts (P < 0.05), which might reflect a defect in cadmium induction of metallothionein in Menkes syndrome fibroblasts, although this might only be an effect resulting from the high basal intracellular copper levels. Thus, cadmium-induced m etallothionein might first be involved in the binding of excess, unlabeled, intracellular copper instead of 64Cu from the medium. This effect may help explain the low three-hour fibroblast 64 Cu activity in tables II, III and IV. As d isc u sse d p rev io u sly, th e relativ ely higher 64 Cu incorporation o f64cu during the first hour of incubation may represent the rapid influx o f64cu, which displaces unlabeled copper and cadmium into the cytosol and culture medium. The lower three-hour64 Cu activity may then reflect the effects of the influx of unlabeled copper back into the fibroblasts. An alternate explanation is that 64Cu displaces cadmium from its binding sites on metallo-

6 ROLE O F M ETALLOTHIONEINS IN PATIENTS W ITH MENKES SYNDROME TABLE IV Effect of Cadmium on Copper Retention in Fibroblasts T im e ( H r s.) MS* cells Skr Cu cpm per mg protein rrr : at 37 C 2.8 Normal cells Cu cpm per mg protein P < 0.05 MS* cells 61*Cu cpm p er mg protein Normal cells b *Cu cpm per mg protein at 37 C in '11 1 presence of cadmium *Menkes kinky hair syndrome. thionein, releasing free Cd++ into the cell cytoplasm or m edium, stim ulating the synthesis of additional m etallothionein, which initially binds intracellular cations, then labeled64 Cu. This hypothesis might also explain the relatively higher64 Cu uptake observed at five hours in tables II, III and IV. Since this effect is not observed in normal fibroblasts, it implies aberration in m etallothionein, perhaps in its metalbinding function. Discussion T he biological function of m etallothionein has not been defined. It has been im plicated in the detoxification of metals, but zinc, an essential trace metal, is its principal constituent and an im portant determ inant of hepatic m etallothionein concentration.5, 10,19,20,30 Factors resulting in zinc red istrib u tio n are re fle c te d in changes in tissue m etallothionein content.30, 31 The uptake of zinc by the liver or small intestine is associated w ith m etallo th io n ein synth esis in those organs.5,12,31,32 Thus, the metallothioneins have been postulated to function in the homeostatic control of total body zinc, as hepatic storage proteins and regulators of intestinal absorption.4,30,31 In animals, orally or parenterally adm inistered cadmium accum ulates principally in the liver and kidneys.18,34,39 Chronic exposure of rats to subtoxic doses of cadmium results in its accumulation in the liver and kidney, b o u n d to m etallothionein.7,21,32 M oreover, after acute exposure of rats to a single dose of cadmium, 75 to 80 percent ofthe dose was found to be bound to hepatic m etallothionein within six hours after the injection.18 T he in creased metallothionein synthesis which occurs coincident with cadmium accumulation has been postulated to indicate its role in cadmium detoxification.22,41 Thus, there are apparently w ell-defined relationships betw een m etallothionein synthesis, cadmium detoxification, and zinc m etabolism. W ith respect to copper m etabolism, however, no clear relationship to m etallothionein has been demonstrated.11 In contrast to zinc or cadmium, m etallothionein synthesis does not occur in response to copper exposure. Instead, copperchelatin has been shown to be synthesized in response to copper challenge and postulated to be the major copper-binding hepatic protein.29,30,41 The m etallothioneins, however, have been implicated in the cellular uptake of copper and in intestinal transport, and there is an abundance of indirect in vivo and in vitro data implying relationships betw een copper and cadm ium or copper and zinc m etabolism i-9' "27 Summary This study in skin fibroblasts dem onstrates an interaction betw een copper and cadm ium m etabolism, w hich M ills suggests may be m ediated in liver and

7 3 0 8 GARNICA, CHAN AND RENNERT kidney through a m etallothionein-type protein.27 Mills adds, however, that the postulated involvement of such proteins in an interaction betw een antagonistic transition elem ents does not presuppose that it m ust occur in liver or kidney, since metallothioneins are present in m ultiple tissues.27 These results reported here, furthermore, suggest alterations of both copper and cadmium homeostasis in the mutant. Menkes syndrome, fibroblasts. Not only do the mutant skin fibroblasts demonstrate abnormalities in copper and cadmium m e tabolism but also in copper-cadm ium interactions. Since the m etallothioneins may be the only cellular mechanism for cadm ium detoxification, these observations implicate, indirectly, an abnormality in metallothionein function, which affects the efflux of copper from the mutant fibroblasts. Whether the observed abnormalities in cadmium metabolism may be an artifact of the high fibroblast copper content cannot be determ ined from this data. Further resolution of this question will require more direct studies. References 1. BREMNER, I.: Copper and zinc proteins in ruminant liver. Trace Element Metabolism in Animals. Hoekstra, W. B., Suttle, J. W., Ganther, H. E., and Merz, W., eds. Baltimore, University Park Press, 1974, pp C a m p b e l l, J. K. and M i l l s, C. F.: Effects of dietary cadmium and zinc on rats maintained on diets low in copper. Proc. Nutr. Soc. 33:15A- 17A, C h a n, W. Y., G a r n ic a, A. D., and R e n n e r t, O. M.: Defective metallothionein and copper accumulation in Menkes Kinky Hair Syndrome fibroblasts. American Society of Human Genetics, 28th Annual Meeting, San Diego, CA, October 19-22, C h e n, R. S., V a s e y, E. J., and W h a n g e r, P. D.: Accumulation and depletion of zinc in rat liver and kidney metallothioneins. J. Nutr. 107: , C h e n, R. W., W h a n g e r, P. D., W e i s i g, P. H.: Biological function of metallothioneins. I. Synthesis and degradation of rat liver metallothionein. Biochem. Med. i2:95-105, D a n k s, D. M., C a m p b e l l, P. E., St e v e n s, B. J., M a y n e, V., and C a r t w r i g h t, E.: Menkes Kinky Hair Syndrome: an inherited defect in copper absorption with widespread effects. Pediatrics 50: , D a v ie s, N. T. and C a m p b e l l, J. K.: The effect of cadmium on intestinal copper absorption and binding in the rat. Life Sci. 20 : , D e k a b a n, A. S., O Re i l l y, S., Aa m o d t, R., and R u m b l e, W. F.: Study of copper metabolism in Kinky Hair Disease (Menkes Disease) and hepatolenticular degeneration (Wilsons Disease) utilizing67 Cu and radioactivity counting in the total body and various tissues. Trans. Amer. Neurol. Assoc. 99: , E v a n s, G. W., M a jo r s, P. F., and C o r n a t z e r, W. E.: Mechanism for cadmium and zinc antagonism of copper metabolism. Biochem. Biophys. Res. Comm. 41: , E v a n s, G. W.: Function and nomenclature for two mammalian copper proteins. Nutr. Rev. 29:195, E v a n s, G. W., W o l n e t z, M. L., and G r a c e, C. I.: Copper binding proteins in neonatal and acute rat liver soluble fraction. Nutr. Reps. Int. 12: , F e l d m a n, S. L. and C o u s in s, R. J.: Degradation of hepatic zinc-thionein after parenteral zinc administration. Biochem. J. 160 : , G o k a, T. J., St e v e n s o n, R. E., H e f f e r a m, P. M., and H o w e l l, R. R.: Menkes Disease. A biochemical abnormality in cultured human fibroblasts. Proc. Nat. Acad. Sci. 73: , G o k a, T. J. and H o w e l l, R. R.: Uptake of radiocopper by cultured skin fibroblasts from individuals with Menkes Disease. American Society of Human Genetics, 28th Annual Meeting, San Diego, CA, October 19-22, H i l l, C. H., M atrone, G., Payne, W. L., and Barber, C. W.: In vivo interactions of cadmium with copper, zinc, and iron. J. N utr.80:227, H o r n, N.: Copper incorporation studies on cultured cells for prenatal diagnosis of Menkes Disease. Lancet/: , H e y d o r n, K., D a m s g a a r d, E., H o r n, N., Mik- KELSEN, M., TYGSTRUP, I., VESTERMARK, S., and WEBER, J.: Extra-hepatic storage of copper. A male fetus suspected of Menkes Disease. Humangenetik 29: , Irons, R. D. and C r ispin-sm it h, J.: Prevention by copper of cadmium sequestration by metallothionein in liver. Chem. Biol. Interact. J5: , Ka g i, J. H. R. and VALLEE, B. L.: Metallothionein: a cadmium and zinc containing protein from equine renal cortex. J. Biol. Chem. 236: , Ka g i, J. H. R., H im m e l h o c h, S. R., W h anger, P. D., Be t h u n e, J. L., and VALLEE, B. L.: Equine hepatic and renal metallothioneins. Purification, molecular weight, amino acid composition, and metal content. J. Biol. Chem. 249: , Kim u r a, M.,O t a k i, N., Yo s h ik i, S., Su z u k i, M., HORIACHI, N., and SlDA, T.: The isolation of metallothionein and its protective role in cadmium poisoning. Arch. Biochem. Biophys. 165: , 1974.

8 ROLE O F M ETALLOTHIONEINS IN PATIENTS W ITH MENKES SYNDROME K o jim a, Y., B e r g e r, C., V a l l e e, B. L., and K a g i, J. H. R.: Amino acid sequence of renal m etallothionein-lb. Proc. Nat. Acad. Sci. 73: , L o w ry,o.h.,r o seb o ro u g h,n.j.,f a rr,a.l., and R a n d a ll, R. L.: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: , M a r g o s h e s, M. and V a l l e e, B. L.: A cadmium protein from equine cortex. J. Amer. Chem. Soc. 79: , M c Ke e, J. E. and W o l f, H. W., eds.: Resources Agency of California. State of California: Water Quality Criteria, 2nd ed. State Water Pollution Control Board, Public 3-A, Sacramento, CA. 26. M i l l s, C. F. and D a l g a r n o, A. C.: Copper and zinc status of ewes and lambs receiving increased dietary concentrations of cadmium. Nature 239: , M il l s, C. F.: Trace element interactions: effects of dietary composition on the development of imbalance and toxicity. Trace Element Metabolism in Animals. Hoekstra, W. B., Suttle, J. W., Ganther, H. E., and Merz, W., eds. Baltimore, University Park Press, 1974, pp NORHEIM, G. and STEINNES, E.: Distribution of some protein bound trace elements among soluble protein fractions from human liver. Acta Pharm. Tox. 38: , P r e m a k u m a r, R., W in g e, D. R., W il e y, R. D., and RAJAGOPALAN, K. V.: Copper-induced synthesis of copper-chelatin in rat liver. Arch. Biochem. Biophys. 170: , R ic h a r d s, M. P. and C o u s in s, R. J.: Isolation of an intestinal metallothionein induced by parenteral zinc. Biochem. Biophys. Res. Comm. 75: , R i c h a r d s, M. P. and C o u s in s, R. J.: Metallothionein and its relationship to the metabolism of dietary zinc in rats. J. Nutr. 106: , R io r d a n, J. R. and G o w e r, I.: Purification of low molecular weight proteins from copper loaded liver. Biochem. Biophys. Res. Comm. 66: , Sc h r o e d e r, H. A., N a s o n, A. P., T ip t o n, I. H. and BALASSA, J. J.: Essential trace metals in man: copper. J. Chron. Dis. i9: , S h a ik h, Z. A., C r is p in - S m ith, J.: The biosynthesis of metallothionein in rat liver and kidney after administration of cadmium. Chem. Biol. Interact. 15: , STARCHER, B. C.: Studies on the mechanism of copper absorption in the chick. J. Nutr. 97: , TERAO, T. and O w e n, C. A.: Effects of copper deficiency and copper loading on67 Cu in supernatants of rat organs. Tohoku ]. Exp. Med. 120: , T e r a o, T. and O w e n, C. A.: Nature of copper compounds in liver supemate and bile of rats: studies with 67Cu. Amer. J. Physiol. 224: , V a n C a m p e n, D. R.: Effects of zinc, cadmium, silver, and mercury on the absorption and distribution of copper-64 in rats. J. Nutr. 88: , W e b b, M.: Binding of cadmium ions by rat liver and kidney. Biochem. Pharm. 21: , W in g e, D. R., P r e m a k u m a r, R., W ile y, R. D., and RAJAGOPALAN, K. V.: Copper-chelatin: purification and properties of a copper-binding protein from rat liver. Arch. Biochem. Biophys. 170: , W in g e, D., K r a g n o, J., and Colucci, A. V.: Cadmium accumulation in rat liver: correlation between bound metal and pathology. Trace Element Metabolism in Animals. Hoekstra, W. B., Suttle, J. W., Ganther, H. E., and Merz, W., eds. Baltimore, University Park Press, 1974, pp