Role of Metallothioneins in Copper Transport in Patients with Menkes Syndrome

ADOLFO D. GARNICA, M.D., W. Y. CHAN, Ph.D.* and OWEN M. RENNERT, M.D.*

Division of Genetics, Department of Pediatrics, University o f Florida College of Medicine, Gainesville, FL 32610 and Department o f Pediatrics, Health Science Center, University of Oklahoma, Oklahoma City, OK 73190

ABSTRACT

Fibroblasts from infants with Menkes kinky hair syndrome, which accumu 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 presumed storage or transport protein metallothionein. Data are presented which are consist ent with defective copper efflux from the mutant cells. Because of the more specific role of metallothionein in cadmium detoxification, studies of cad mium metabolism were undertaken which demonstrated abnormal cadmium retention and metallothionein 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 metallothionein in copper transport for fibroblasts.

Introduction tute significant components. Metallothio nein synthesis in response to metal loading The metallothioneins are zinc, copper in animals implicates them in heavy metal and cadmium-binding intracellular pro detoxification and/or essential trace metal teins of low molecular weight, which con m etabolism .5, 10, 21, 28,30 M etallothionein tain 30 to 35 percent cysteine and 6 to 11 synthesis occurs in response to toxic metal percent metal bound to cysteinyl side challenge. However, the liver content of chains by mercaptide bonds. They occur in zinc, an essential trace metal, has been multiple tissues in some species, in forms reported to be an important determinant of of varying amino acid composition, de the concentration of metallothionein-type spite which their biological function has not proteins.1,5,11,31 been established.20,22 In human liver, zinc The function of metallothionein in in is the principal metal constituent, al tracellular copper transport was suggested though copper and cadmium also consti by the identification and isolation of a

302 0091-7370/78/0700-0302 $01.20 © Institute for Clinical Science, Inc. ROLE OF METALLOTHIONEINS IN PATIENTS WITH MENKES SYNDROME 3 0 3 cytosol protein of an estimated 10,000 Amersham/Searle. 64 Cu and 109 Cd were molecular weight which apparently is counted in a gamma counter.* Metal involved in intestinal copper absorp analysis was performed with an atomic tion.9, 10,36,37 A copper-binding cytosol pro absorption spectrophotometerf equip tein of molecular weight 11,000, the same ped with graphite furnace. Protein was estimated size as metallothionein, has determined by the method of Lowry been implicated in the cellular uptake of et a l.23 copper.36,37 On the basis of similarities be tween this protein fraction and the metal- C o p p e r -D o s e R e s p o n s e lothioneins, a role has been postulated for metallothionein in intracellular copper Copper sulfate was added to confluent transport.9,10,37 fibroblast cultures to achieve copper con Copper is potentially toxic to all forms centrations of a 2 to 40 ju.g per ml. The of life, especially those having no capac cultures were incubated for 48 hours at ity to limit its uptake. In such instances, 37° C, trypsinized and homogenized, and susceptibility to copper toxicity, of the supernatant copper and protein necessity, becomes a function of cellular measured. excretory efficiency. The viability of cul tured fibroblasts, despite their permea C a d m iu m C o n t e n t bility to copper, thus implies the function of efficient homeostatic mechanisms, and Cadmium chloride was added to basal the accumulation of excessive amounts of culture medium to achieve cadmium copper by skin fibroblasts from individu concentrations of 0.1 to 1.1 meg per ml. als with Menkes kinky hair syndrome Confluent fibroblast cultures were incu might represent a failure of such mecha bated for 48 hours at 37°C in medium of nisms.6,13,14,16 This report reviews studies increasing cadmium concentration, then attempting to clarify the molecular basis harvested, homogenized and the super of this abnormality and the role of metal natant protein and cadmium determined. lothionein in its pathogenesis. A decrease in copper efflux from Menkes syndrome C a d m iu m a n d C o p p e r U p t a k e fibroblasts is recorded, along with an aberrant response to cadmium challenge, Precisely 17 ju.Ci o f64Cu were added to which implies an inborn defect in copper confluent cultures in 10 ml of medium, and cadmium metabolism. These obser the Cu++ concentration of which was ad vations support the intracellular detoxifi justed to 9 /xg per ml with CuS04. Cad cation and transport functions postulated mium, if present, was added to a concen for metallothionein.5,1 u21,32,35 tration of 0.09 fig per ml. The cultures were incubated at 37° C for one to 10 hours, harvested, homogenized, the pro Materials and Methods tein content of the lysate determined and Skin fibroblast cultures were explanted the 64Cu counted. 109 Cd uptake by cul from three infants w'ith Menkes kinky tured fibroblasts were examined at 37°C. hair syndrome. Two additional mutant Four nCi of109 Cd were added to the cul cultures and control cultures were ob ture medium and the Cd++ concentration tained from the Human Genetic Cell Re adjusted to 0.56 ng per ml with CdCl2. pository and the American Type Culture Collection. 64Cu was obtained from the * Packard Model S210. New England Nuclear and 109Cd from t Perkin-Elmer Model 306. 304 GARNICA, CHAN AND RENNERT

C o ppe r E f f l u x concentrations of 10 through 15 meg per ml, the copper content of both cultures in Cultures were pulse-labeled with 16.7 creased above the baseline levels, while ju.Ci of 64Cu at 37°C for 20 hours, then that of the Menkes syndrome cells re transferred to non-radioactive medium. mained greater than the normal (P < Cultures were harvested and analyzed for 64 Cu and protein one to 10 hours after the 0.04). At medium concentrations equal to or above 20 meg per ml, the copper con medium change. tent of the normal cells was not signifi Statistical differences were examined cantly different from that of the mutant for significance using a t-test for paired cells. data. These observations suggest that copper Results uptake by the mutant fibroblasts is at least equal to that of normal fibroblasts. F ib r o b l a st C o p p e r a n d C adm ium Furthermore, they imply that the copper C o n t e n t regulatory mechanisms in normal fibro Cultured Menkes syndrome fibroblasts blasts function effectively up to a medium are morphologically normal but have a copper concentration of approximately 15 diminished tolerance to copper.4 The in meg per ml, while those of Menkes syn creased sensitivity of these cells to cop drome fibroblasts are abnormal at all con- per toxicity is associated with increased concentrations. An inconsistency in the intracellular copper content.13,16 In basal general relationship between medium cop culture medium (copper concentration per concentration and fibroblast copper 48.70 ± 20.0 ng per ml) the copper con content involving both cell lines is appa tent of normal cells was 108 ng per mg rent at a medium concentration of 1 meg cell protein, while that of the mutant fi per ml, which most plausibly may be ex broblasts was 310 ng per mg (table I). As plained in terms of a ten-fold error in di the copper concentration of the medium lution, since the values at one and 10 meg was gradually increased, the copper con per ml are essentially the same for both tent of neither cell culture increased sig cultures. nificantly until the medium copper con As the cadmium concentration of the centration exceeded 6 meg per ml. At culture medium was increased from basal

T A B L E I

Copper and Cadmium Content of Cultured Skin Fibroblasts

Medium (Cu++) Copper Content*+ Medium (Cd++) Cadmium Content *§ meg p e r ml MS* (n=7) N orm al (n=4) m eg p e r ml MS (n-5) Normal (n=4)

+ + 0 3 1 0 + 6411 1 0 8 ± 2 1 0 2 3 . 6 6.4 1 ! 2 3 . 5 93 + 1 6 0 5 + 1 8 9 2 2 2 + 74 0 . 1 1 9 2 4 4 1 8 0 ± 8 4 + + 4 2 5 3 + 4 6 1 0 6 + 2 0 0 . 3 8 6 6 3 9 2 2 4 1 4 9 + + 6 2 6 4 ± 5 0 1 5 4 + 37 0 . 5 1 1 6 5 2 1 6 6 2 0 2 0 7 1 0 6 3 7 + 2 2 9 2 5 9 + 94 0 . 7 1 0 7 3 + 3 6 3 7 6 5 ± 93 + + 15 8 0 4 + 1 1 1 4 5 2 + 1 3 3 0 . 9 1 4 3 4 1 5 7 8 0 1 1 1 3 + 2 0 9 7 5 + 3 3 1 1 0 8 0 + 1 5 1 1 . 1 1 0 1 8 + 2 2 8 6 5 5 1 2 3 25 1 1 3 9 + 1 9 5 1 9 1 3 + 6 0 8 30 1 3 6 7 + 332 1 5 5 3 + 383 35 2 1 5 0 + 1 0 1 2 2 0 8 6 + 5 1 6 40 2 2 2 2 + 4 1 8 2 4 7 5 + 7 2 0

*Units expressed as ng metal per mg cell protein. tP < 0.04 §P < 0.05 xMenkes kinky hair syndrome. liValues expressed are means ± standard deviation. ROLE OF METALLOTHIONEINS IN PATIENTS WITH MENKES SYNDROME 305 levels, so did the cadmium content (ng TABLE I I Cd per mg cell protein) of the normal and Copper-64 and Cadmium-109 Incorporation by Skin Fibroblasts at 37°C Menkes syndrome fibroblasts (table I).

They remained essentially equal, how Time &Vopper*t§ 109Cadjniuin*/íF ever, up to a medium concentration of 0.3 Hrs. MS* (n=3) Normal (n=4) MSX (n=3) Normal (n=3) meg per ml, when the cadmium content 0 of the mutant cell line increased sharply. 1 7.0 ± 0.7§ 2.5 ± 0.7 8.6 ± 0.61 7.7 ± 0. 3 3 2.2 ± 0.3 3.5 ± 0.7 9.6 ± 0. 6 7.4 ± 0.3 This did not occur in the normal culture 5 8.9 ± 0.5 2.2 ± 0.5 10.3 ± 1.6 5.9 ± 0.0 7 5.4 ± 0.2 2.0 ± 0.2 12.6 ± 0.3 7.4 ± 0.3 until the medium concentration reached 10 5.5 ± 0.2 2.4 ± 0.6 13.8 ± 4.0 7.1 ± 0.6 0.5 meg per ml. At medium concen *10~:i x 61+Cu cpm per mg cell protein or 109Cd cpm per mg trations of 0.5 to 1.1 meg per ml the fi cell protein. broblast cadmium content of both cultures tSpecific activity &4Cu, 910 uCi per mg. §P < 0.05 stabilized, with the cadmium content of ^Values expressed as means ± standard deviation. 1TP < 0.02 the mutant fibroblasts remaining greater *Menkes kinky hair syndrome. than that of the normal fibroblasts (P < 0.05). is followed by a rapid shift intracellularly of the displaced copper and, finally, re C o p p e r a n d C a d m iu m U p t a k e equilibration and stabilization. In all but In order to investigate further the the third hour, 64Cu incorporation was mechanism underlying the high copper consistently equal to or greater than into content of Menkes syndrome fibroblasts, mutant fibroblasts than into normal fi the uptake of radioactive copper and broblasts (P <0.05). These results substan cadmium was measured. A relatively tiate the observation that copper influx high medium copper concentration was into Menkes syndrome fibroblasts is not used to maximize the differences in cop decreased and are consistent with reports per homeostasis between the two cell of copper uptake and absorption, which types, but it was maintained sufficiently indicate that the uptake of copper into in below cytotoxic levels to avoid artifac- testinal mucosal cells and other tissues of tual values resulting from cell injury. patients with Menkes syndrome is in During the ten-hour observation period, tact.6'8,14,17 Assuming that the removal of the 64 Cu activity of normal skin fibro copper from cultured fibroblasts is, to blasts did not vary significantly, possibly some degree, a function of intracellular reflecting a stable equilibrium between copper concentration, these observations 64Cu uptake and discharge (table II). The suggest that the copper excretory mecha 64Cu activity of Menkes syndrome fibro nisms of Menkes syndrome fibroblasts blasts, however, did not become stable function less effectively than those of until after seven hours at a level approx normal cells. imately twice normal. The 64 Cu activity Incubation of normal fibroblasts with of the mutant fibroblasts at one and five 109 Cd-containing medium demonstrated a hours was two to three times that of the constant, stable level of 109Cd-radioac- normal fibroblasts. At three hours, how tivity throughout the study period (table ever, the 84Cu activity in Menkes syn II). The109 Cd radioactivity retained by the drome fibroblasts was approximately Menkes syndrome fibroblasts, however, equal to that of the normal fibroblast. was cons istently higher than in the normal This observation might be explained in fibroblasts and increased with incubation terms of a rapid influx of 64 Cu into the time (P < 0.02), corroborating the abnor Menkes syndrome cells driven by a con mality in transport or storage implied by centration gradient, which displaces un the increased cadmium content of the labeled copper during the first hour. This mutant cells (table I). 3 0 6 GARNICA, CHAN AND RENNERT

C o p p e r R e t e n t io n C o p p e r -C a d m iu m I n t e r a c t io n To clarify whether the defect in Menkes In vivo animal studies have demon syndrome fibroblasts involved copper up strated an interaction between cadmium take or excretion,64 Cu efflux was studied and copper m etabolism .2,3’15,18,25,26 by the pulse-labelling of cultured cells. Simultaneous administration of cadmium During this phase of the study, skin fibro and copper salts to rats results in a decrease blasts were first grown in64Cu-containing in cadmium sequestration by metallo- medium, which was then replaced by un- thionein and an increase in cadmium labeled medium, the cells harvested se toxicity.18,26 Assuming, therefore, a role rially after transfer and counted for 64 Cu for the metallothioneins in fibroblast cop activity. Any defect in efflux would thus per metabolism or homeostasis,64 Cu up appear as an increase in retained radioac take or incorporation might be expected to tivity. At 37°C, 64Cu retention was greater be influenced by the presence of cad in the mutant than in the normal fibro mium in the culture medium. To blasts, except during the first and third minimize the effects of variation in ex hours after transfer to non-radioactive perimental conditions, the influence of medium (table III). At one and three cadmium on copper retention is ex hours, the retention of64 Cu by normal and pressed as the ratio o f64Cu incorpor mutant cells was essentially equal. At five ation in Menkes syndrome: normal hours and beyond, however,64 Cu reten fibroblasts. tion in the Menkes syndrome fibroblasts In the presence of 0.09 ng per nl of was greater than in normal cells (P < cadmium, copper uptake by both fibro 0.025). Since the Menkes syndrome and blast lines is increased (table IV). 64 Cu normal cultures were exposed to non uptake, however, is increased to a greater radioactive basal medium during the degree in normal fibroblasts than in chase, any difference in 64 Cu retention Menkes syndrome fibroblasts (P < 0.05), may be attributed to differences in copper which might reflect a defect in cadmium efflux. Thus, at 37°C, the Menkes syn induction of metallothionein in Menkes drome cells demonstrate evidence of im syndrome fibroblasts, although this might paired copper efflux. only be an effect resulting from the high basal intracellular copper levels. Thus, cadmium-induced metallothionein might T A B L E III first be involved in the binding of excess, Copper-64 Retention by Cultured unlabeled, intracellular copper instead of Fibroblasts at 37°C*+ 64Cu from the medium. This effect may help explain the low three-hour fibroblast Time o f Chase (Hrs.) MS* (n=4) Normal (n=4) 64 Cu activity in tables II, III and IV. As discussed previously, the relatively 0 7346 ±1726§ / 4670 ± 1430 higher 64 Cu incorporation of64Cu during 1 23.4 ± 8.0 / 25.7 ± 5.1 3 19.1 ± 5.5 19.1 ± 2.7 the first hour of incubation may represent 5 30.3 ±4.0 17.8 ± 4.1 the rapid influx of64Cu, which displaces 7 27.0 ± 5.4 11.6 ± 2.5 10 20.9 ± 6.0 12.2 ± 4.0 unlabeled copper and cadmium into the cytosol and culture medium. The lower ♦Specific activity of cl+Cu, 595 pCi per mg. three-hour64 Cu activity may then reflect tP < 0.025 §Units expressed, ^4Cu cpm per mg cell protein, the effects of the influx of unlabeled cop /values expressed as means ± standard deviation. per back into the fibroblasts. An alternate U/.fter zero time, values expressed are percentages of zero time counts. explanation is that 64Cu displaces cad xMenkes kinky hair syndrome. mium from its binding sites on metallo- ROLE OF METALLOTHIONEINS IN PATIENTS WITH MENKES SYNDROME 307

TABLE IV

Effect of Cadmium on Copper Retention in Fibroblasts

Time (Hrs.)

MS* cells Skr Cu cpm per mg protein “------rrr“------— :— at 37°C 2.8 Normal cells ° Cu cpm per mg protein7 P < 0.05 MS* cells 61*Cu cpm per mg protein Normal cells b “*C u cpm per mg protein at 3 7 °C in 1-2 0-8 0-9 1'1 1 •1 presence of cadmium

*Menkes kinky hair syndrome. thionein, releasing free Cd++ into the cell lothionein.7,21,32 Moreover, after acute ex cytoplasm or medium, stimulating the posure of rats to a single dose of cadmium, synthesis of additional metallothionein, 75 to 80 percent ofthe dose was found to be which initially binds intracellular cations, bound to hepatic metallothionein within then labeled64 Cu. This hypothesis might six hours after the injection.18 The in also explain the relatively higher64 Cu up creased metallothionein synthesis which take observed at five hours in tables II, III occurs coincident with cadmium accumu and IV. Since this effect is not observed in lation has been postulated to indicate its normal fibroblasts, it implies aberration in role in cadmium detoxification.22,41 Thus, metallothionein, perhaps in its metal- there are apparently well-defined rela binding function. tionships between metallothionein syn thesis, cadmium detoxification, and zinc Discussion metabolism. With respect to copper metabolism, The biological function of metallothio however, no clear relationship to metal nein has not been defined. It has been lothionein has been demonstrated.11 In implicated in the detoxification of metals, contrast to zinc or cadmium, metallothio but zinc, an essential trace metal, is its nein synthesis does not occur in response principal constituent and an important de to copper exposure. Instead, copper- terminant of hepatic metallothionein con chelatin has been shown to be synthesized centration.5, 10,19,20,30 Factors resulting in in response to copper challenge and post zinc redistribution are reflected in ulated to be the major copper-binding changes in tissue metallothionein con hepatic protein.29,30,41 The metallothio- tent.30, 31 The uptake of zinc by the liver or neins, however, have been implicated in small intestine is associated with metal the cellular uptake of copper and in intes lothionein synthesis in those or tinal transport, and there is an abundance gans.5,12,31,32 Thus, the metallothioneins of indirect in vivo and in vitro data imply have been postulated to function in the ing relationships between copper and homeostatic control of total body zinc, as cadmium or copper and zinc metabo hepatic storage proteins and regulators of lism i-9'15-18-26"27 intestinal absorption.4,30,31 In animals, orally or parenterally ad Summary ministered cadmium accumulates princi pally in the liver and kidneys.18,34,39 This study in skin fibroblasts demon Chronic exposure of rats to subtoxic doses strates an interaction between copper and of cadmium results in its accumulation in cadmium metabolism, which Mills the liver and kidney, bound to metal suggests may be mediated in liver and 3 0 8 GARNICA, CHAN AND RENNERT kidney through a “metallothionein-type” copper absorption with widespread effects. protein.27 Mills adds, however, that the Pediatrics 50:188-201, 1972. 7. D a v ie s , N. T. and C a m p b e l l , J. K.: The effect of postulated involvement of such proteins in cadmium on intestinal copper absorption and an interaction between antagonistic transi binding in the rat. Life Sci. 20 :955-960, 1977. tion elements does not presuppose that it 8. 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 must occur in liver or kidney, since metal- Kinky Hair Disease (Menkes Disease) and lothioneins are present in multiple tis hepatolenticular degeneration (Wilsons Dis ease) utilizing67 Cu and radioactivity counting in sues.27 These results reported here, further the total body and various tissues. Trans. Amer. more, suggest alterations of both copper Neurol. Assoc. 99:106-109, 1974. and cadmium homeostasis in the mutant. 9. 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 an Menkes syndrome, fibroblasts. Not only tagonism of copper metabolism. Biochem. do the mutant skin fibroblasts demonstrate Biophys. Res. Comm. 41:1142-1148, 1970. abnormalities in copper and cadmium me 10. E v a n s , G. W .: Function and nomenclature for two mammalian copper proteins. Nutr. Rev. tabolism but also in copper-cadmium in 29:195, 1971. teractions. Since the metallothioneins 11. E v a n s , G. W ., W o l n e t z , M. L., and G r a c e , C. may be the only cellular mechanism for I.: Copper binding proteins in neonatal and acute rat liver soluble fraction. Nutr. Reps. Int. cadmium detoxification, these observa 12:261-269, 1974. tions implicate, indirectly, an abnormality 12. F e l d m a n , S. L. and C o u s in s , R. J.: Degradation in metallothionein function, which affects of hepatic zinc-thionein after parenteral zinc the efflux of copper from the mutant fibro administration. Biochem. J. 160 :583-588,1976. 13. 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. blasts. Whether the observed abnormalities M., and H o w e l l , R. R.: Menkes Disease. A in cadmium metabolism may be an artifact biochemical abnormality in cultured human fi of the high fibroblast copper content can broblasts. Proc. Nat. Acad. Sci. 73:604-606, 1976. 14. G o k a , T. J. and H o w e l l , R. R.: Uptake of not be determined from this data. Further radiocopper by cultured skin fibroblasts from resolution of this question will require individuals with Menkes Disease. American Society of Human Genetics, 28th Annual Meet more direct studies. ing, San Diego, CA, October 19-22, 1977. 15. H i l l , C. H ., M atrone, G., Payne, W. L., and Barber, C. W.: In vivo interactions of cadmium References with copper, zinc, and iron. J. Nutr.80:227,1963. 16. H o r n , N.: Copper incorporation studies on cul 1. BREMNER, I.: Copper and zinc proteins in ru tured cells for prenatal diagnosis of Menkes Dis minant liver. Trace Element Metabolism in ease. Lancet/:1156-1157, 1976. Animals. Hoekstra, W. 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