Studies on the zinc content of Cd-induced thionein

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 188, No. 2, June, pp. 466-475, 1978 Studies on the Zinc Content of Cd-lnduced Thionein D E N N I S R. WINGE, 2 R. P R E M A K U M A R , 3 AND K. V. R A J A G O P A L A N Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Received December 14, 1977; revised February 6, 1978 Liver metallothionein can be induced by the injection of a variety of metal ions, including Cd, Hg, Ag, Au, and Zn, into rats. The protein so induced contains bound zinc in addition to the other metal ion used for induction. In Cd-induced metailothionein Zn ~§ was found to occupy two of the eight possible metal-binding sites in thionein prepared from animals killed at least 10 h after the injection of cadmium. Thion'ein isolated at earlier times contained little or no zinc. The amount of thionein formed is proportional to the dose of Cd, but the hepatic uptake of Cd precedes the mobilization of Zn by several hours. This Cdinduced mobilization of Zn is most significant in the liver. The origin of the mobilized Zn is primarily from the excretory pathway, with the diet also being a contributing factor. In mice and rats, the increased hepatic Zn arising from Cd injection can largely be accounted for by a decrease in fecal Zn. This effect of Cd-induced Zn mobilization is also observed in rats and mice made zinc deficient. The mechanism of Zn mobilization is not resolved, but may involve both an effect of cadmium on excretion and the retention of zinc in the liver caused by an elevated level of thionein. The results presented suggest a physiological role for thionein in zinc metabolism. Metallothionein, a low molecular weight cytoplasmic protein with a high cysteine content, was first described by Margoshes and Vallee in 1957 (1) and has since been characterized from a variety of animal sources (2-9). The amino acid sequence of the equine renal thionein has recently been determined by Kojima et al. (10). In addition to similarities in amino acid composition and molecular weight, all metallothioneins isolated from animal tissues to date have exhibited polymorphism and have been shown to contain zinc. When metallothionein is induced in rats by any of a variety of metal ions, including cadmium, mercury, and silver, the isolated protein invariably contains zinc in addition to the inducing metal (2). In each case the hepatic zinc content is elevated above control values, and this increased liver zinc level is reflected in the thionein zinc content. The 1This work was supported by Grant GM 00091 from the U.S. Public Health Service. ') Recipient of National Institutes of Health Postdoctoral Fellowship ES 07002. Present address: Department of Biology, McMaster University, Hamilton, Ontario, Canada. 466 0003-9861/78/1882-0466502.00/0 Copyright (D 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. mechanism by which hepatic and thionein zinc levels increase in response to the administration of a metal ion such as cadmium or mercury has not been explained. This paper describes studies on the identification of the sources and factors involved in the Cd-induced zinc mobilization in rat and mouse. MATERIALS AND METHODS Chemicals and gels were obtained as described previously (2). 65Zn was purchased from New England Nuclear and used at a specific activity of 1 Ci/g. Male Sprague-Dawley rats (CD strain) from Charles River Laboratories and male Swiss Webster mice from Hilltop Laboratories were used in these studies. The animals were maintained on normal Purina Rat Chow or on special diets (Zn-deficient or normal-protein test diets} obtained from Teklad. The Zn-deficient diet was found to contain 0.5 pg of zinc/g of diet. The animals were housed in plastic cages except in metabolic experiments, where specially designed stainless-steel cages were used. Metal salts were administered by a variety of routes, including subcutaneous or intraperitoneal injection and oral feeding by stomach intubation. Metallothionein was purified as described previously from liver and kidney (2). The protein concentration of the purified thionein was determined by dry weight anal- 467 ZINC CONTENT OF Cd-INDUCED THIONEIN ysis after exhaustive dialysis in deionized water. The protein was dried to a constant weight in a forced-air oven and weighed on a Cahn electrobalance from Ventron Instrument Co. Metal analysis was performed using a Perkin-Elmer Model 107 flame atomic absorption spectrometer. Prior to analysis, tissues were ashed in a muffle furnace before or after homogenization followed by resuspension in 0.5 N HCI. Complete extraction of metal ions in fecal matter required wet ashing in HCI:HNO3 (3:1) after dry ashing. ~Zn was measured using an Intertechnique Model CG30 gamma spectrometer. Sedimentation equilibrium measurements were carried out on a Beckman-Spineo Model E analytical ultracentrifuge equipped with a photoelectric scanner. Absorbance at 250 nm was used to measure protein concentration as a function of position in the cell. A partial specific volume of 0.64 was determined experimentally with a Precision CMA 02-C density meter according to the procedure of Kratky et al. (ll). RESULTS We previously demonstrated that a variety of metal ions have the ability to induce thionein formation (2). The metal ions reported in that study included cadmium, mercury, silver, and zinc. One aspect in common for the former three ions was that each either directly or indirectly altered the normal zinc metabolism in such a way as to produce a marked increase in the hepatic zinc content. The rise in liver zinc could largely be accounted for by the increased zinc binding of thionein. In an attempt to determine whether other metals could induce similar changes in zinc metabolism, a variety of ions, including AuCh, SnCl2, PdC12, Pb(NOz)2, and CuC12, was tested. Separate pairs of rats were treated with the individual metal ions by injection at a dose of 2.5 mg of metal ion/kg of body weight, twice over a period of 2 days. Sixteen hours after the second injection, the animals were killed. Determination of the zinc content of the liver suggested that auric chloride (AuCh) was the only compound tested that elicited a positive response. Gel filtration of the liver supernatant fraction showed that the mobilized zinc was bound to a thioneinlike component. The same two polymorphic forms of thionein observed after induction with other metal ions were observed after DEAE4-cellulose chromatography of the gold-induced protein fraction {Fig. 1). Pre4 Abbreviation used: DEAE, diethylaminoethyl. sumably the two thioneins also contained bound gold, but such analysis could not be carried out. The fact that gold, as well as cadmium, mercury, and silver ions, does alter normal zinc metabolism, whereas several other ions do not, suggests that the effect is somewhat specific. To investigate the metal-induced zinc mobilization, cadmium was selected as the inducing metal ion, because of its ability to induce much greater amounts of thionein than the levels induced by the other ions mentioned. Pure Cd-induced metallothionein from various liver preparations was found to contain 5.8 g-atoms of cadmium and 2.0 g-atoms of zinc per mole. In these calculations, the protein was determined by dry weight analysis and the molecular weight by sedimentation equilibrium. Sedimentation studies were performed on the purified protein in nondenaturing solvents. Plots of In concentration versus the square of the distance from the center of rotation displayed good linearity at each of three speeds, indicative of a homogeneous protein preparation. The molecular weight calculated from seven independent measurements using an experimentally derived partial specific volume of 0.64 was found to be 8400 _+ 370. This weight-average molecular weight represents the weight of the // Io., I 0.8 E o.6 ~ i 0.4 U ~'N I [ I IO 20 30 I ~ l 40 50 60 Froctiom Number 0.2 I 70 80 FIG. 1. DEAE-eellulosechromatographyofAuthionein. Au,Zn thionein, isolated from G-75 column chromatography of the soluble protein fraction from livers of rats injected with AuCt3, was dialyzed against 10 mM Tris-Cl, pH 8.6, and applied to a DEAE-cellulose column (4 x 1.9 cm) equilibrated with the same buffer. The column was washed with 150 ml of the same buffer at a flow rate of 30 ml/h followed by elution with a linear gradient of 0-0.5 M NaCl. Elution fractions of 6.5 ml were collected and monitored for Zn and conductivity. 468 WINGE, PREMAKUMAR, AND RAJAGOPALAN metal-protein complex. '~ The zinc mobilization is not restricted to the liver, but we chose to study the hepatic uptake process because of its greater magnitude than that in other tissues. It was found that the increase in the total hepatic zinc content was directly related to the injection dose of CdC12 at doses of the metal below 2 mg of Cd/kg (Fig. 2). The animals used in this experiment were pretreated with two subcutaneous injections of ~Zn (20-#Ci total) 3 days prior to the single subcutaneous injection of CdCl2. There were corresponding increases in the total zinc and in the ~Zn content of liver. In a related experiment, it was observed that increasing doses of CdCl2 given orally led to corresponding rises in liver zinc and cadmium. Thus, regardless of the route of cadmium administration, the liver zinc content increased with increasing doses of cadmium. The kinetics of uptake of cadmium and zinc did not show parallel behavior. Rats prelabeled with two subcutaneous injections of 65Zn were given a single subcutaneous injection of CdC12 (2.5 mg of Cd/kg) 4 days after the second 6SZn injection, and groups of three animals were killed at varying times. As can be seen in Fig. 3, the liver uptake of cadmium preceded the uptake of zinc by several hours. The slight depression in liver zinc seen at early times after cadmium injection was reproducible and has been observed by other investigators {12). It appeared from this experiment that zinc uptake occurred after a lag period. To substantiate this observation, rat liver metallothionein was isolated by Sephadex G-75 column chromatography 2 or 3 h after the injection of CdC12 into the animals. After the liver extract was heated at 60~ for I0 rain and centrifuged, the clear supernatant was fractionated by gel filtration. Figure 4 shows the elution pattern from the 2-h injection experiment. Approximately 45% of the soluble cadmium eluted in the thionein region, with the remaining amount eluting in an uncharacterized fraction of higher molecular weight. At 3 h the percentage of metal in the thionein fraction had risen to "~Winge, D. R., Premakumar, K., Schechter, N. M., and Rajagopalan, K. V., unpublished observation. i.o 20 J.o q,o FIG. 2. Hepatic zinc uptake as a function of the injection dose of CdCI2. Rats pretreated with ~Zn were injected subcutaneously with saline or CdC12 at the doses shown. The animals received two such injections on the third day after the ~Zn prelabeling. The rats were killed 16 h after the second CdCl2 injection. Cadmium (--4b--), zinc (--A--), and 65Zinc ( ~ ) were analyzed in crude fiver homogenatas. The values shown are averages of three animals per group, with variability less than 15%. 4O I j- .,T 6I I 118 12 H~Jrl P(~t ~ljectiofl ~4 ~0,000 FIG. 3. Rate of liver uptake of Cd and Zn after a single injection of CdCl2. The livers of rats treated as described in the text were homogenized in 5 vol of 0.01 M potassium phosphate, pH 7.8, at 4~ Each point represents the average of three animals, with variability less than 12%. about 65%, with a corresponding decrease in the amount present in the more excluded volume. In both experiments, the thionein fractions were virtually devoid of zinc, suggesting that the mobilization of zinc and incorporation are not essential for thionein induction. Liver thionein formed within a few hours after cadmium administration has a molar Cd/Zn ratio of 8 or greater, whereas thionein isolated after 12 h invariably displays a ratio of 2 to 3. In contrast, renal thionein formed 12 h after cadmium injection usually exhibits a Cd/Zn molar ratio between 6 and 7. The hepatic and ZINC CONTENT OF Cd-INDUCED THIONEIN 0.6 O.2 JO 20 30 40 50 60 70 80 9O ~00 ~tO F~ctJon ~ b e r FIG. 4. Sepbadex G-75 column chromatography of the liver-soluble protein fraction of rats injected with CdCle 2 h prior to killing. The heat-treated cellular supernatant from three rat liver homogenates was chromatographed on Sephadex G-75 (85 x 4 cm) equilibrated with 0.01 M potassium phosphate, pH 7.8, at 4~ The flow rate was 25 ml/h, and 6.5-ml fractions were collected. renal Cd/Zn ratios remain relatively constant with time. In an experiment where six groups of rats were given a single injection of CdCl~ (2.5 mg/kg) and killed 1 to 6 months thereafter, it was observed that the Cd/Zn ratios of metallothionein isolated from the livers and kidneys after 6 months corresponded to the ratios seen at 1 day.6 The source of the zinc which is mobilized to the liver and kidney has not been adequately investigated. In an attempt to elucidate the factors involved, studies were performed on rats labeled with S~Znprior to cadmium administration. The animals were killed 16 h after the second cadmium injection and various tissues were excised. As can be seen in Table I, Cd treatment resulted in varying extents of increased 6~Zn uptake or retention in several tissues, the greatest increase being in liver. Although the increased radioactivity could reflect greater uptake or tissue retention, the slow turnover of zinc in bone (13) suggests that the change must be a result of enhanced uptake. If tissue uptake of zinc is facilitated, then zinc must be made available from a body pool or from dietary sources. In an attempt to determine the extent of the dietary contribution, ~Zn was administered to rats by force-feeding either 3 h before or 3 h after a single injection of CdCl~. It was found that cadmium caused a three-fold'increase in 65Zn uptake into the liver, regardless of whether the former was injected before or Ridlington, J. W., Winge, D. It., and Fowler, B. M., unpublished observation. 469 after the ~Zn intubation. Further, the dietary 65Zn was demonstrated to be incorporated into hepatic thionein, showing that dietary zinc can contribute to the zinc content of thionein. However, the diet cannot be the major source of zinc, since animals starved 24 h prior and 16 h subsequent to the cadmium injection still showed substantial hepatic zinc mobilization. The exclusion of diet as the major reservoir of mobilized zinc left the body pools as likely sources. To determine whether a highly mobile pool of zinc existed in the body, it would be critical to analyze the whole animal. Since a whole body radioactivity counter was unavailable, mice were selected as the experimental animal, as they could be conveniently counted in a normal gamma spectrometer. Initial experiments demonstrated that cadmium induced a similar mobilization of zinc in mice as in rats and that the mobilized zinc was incorporated into thionein. In the whole body studies, mice were given three subcutaneous injections of 65Zn (30-#Ci total) and after 3 days were given two subcutaneous injections of CdCl2 (2.5 mg of Cd/kg) on successive days. The animals were killed 24 h following the second cadmium injection. Control and Cd-treated mice were dissected into similar parts and counted. Table II shows the results of the total animal counting. The 6~Zncontent was enhanced in several body parts of the Cdtreated mice, with the liver again showing the most pronounced uptake both in percentage and in absolute amount. No major reduction in eSZn was observed in any tissue or body segment, alone or in summation, to account for the substantial elevation in hepatic zinc. In the above experiment the animals were not housed in metabolic cages, so excretory material was not analyzed. The experiment was repeated with the mice maintained in stainless-steel metabolic cages. In this second study, the mice were given two ~Zn preloads followed by a single injection of CdC12 3 days later and were killed 7 days after the Cd treatment. The e~Zn uptake was similar to the data in the previous experiment, with the liver content increasing by over 100% (Table III). Fecal matter 470 WINGE, PREMAKUMAR, AND RAJAGOPALAN TABLE I METAL CONTENT OF RAT TISSUES FOLLOWING CADMIUM INJECTION a Tissue Liver Kidney Heart Spleen Lung Tibia Cd (tLg/g) Zn (#g/g) ~Zn (cpm/g) Control Cd treated Control Cd treated Control Cd treated 0.5 0.2 0.6 0.5 0.3 0.5 47.2 7.1 3.0 4.9 1.6 2.5 23.8 16.8 18.5 18.6 15 126 37.8 18.6 18.5 19.5 15 123 34,000 24,300 24,500 24,400 21,500 65,500 54,000 29,000 27,700 29,000 21,500 72,500 ~'Animals were pretreated for 5 days with 5 tLCiof~Zn (0,7 mg of Zn/kg) prior to two subcutaneous injections of CdCl2 (2.5 mg/kg) or isotonic saline. The rats were killed 16 h following the second injection of CdCl2. The values shown are averages of two, and the duplicates agreed within 15% variation in all eases. TABLE II Cd-INDUCED MOBILIZATIONOF ZINC IN THE MOUSE" Tissue 65Zn (cpm/tissue) Control TABLE III EFFECT OF Cd ON 65Zn EXCRETION IN THE MOUSE" Tissue 6~Zn (cpm/tissue) Cd treated Liver 157,000 + 2,600 309,500+ 35,800 Soft organs ~ 60,700 • 8 , 5 0 0 77,500• 9,500 Intestines' 236,700+ 31,500 257,100+ 24,300 Head 481,150 +_ 26,500 474,300 + 61,600 Front legs 200,000 • 8,000 200,600+ 12,500 Rib cavity 313,500• 66,200 301,900• 36,300 Total body 183,400• 40,300 235,900+ 37,200 hair Rear legs 136,800 • 21,200 140,900• 4,600 Tail 167,700 • 7,500 184,200• 29,200 Remaining 174,000+_ 18,500 152,900• 13,609 carcass "Six mice were pretreated with ~Zn as described in the text. Three animals were injected subcutaneously with CdCl2 (2.4 mg of Cd/kg). The mice were dissected into similar parts and counted in a gamma counter. All parts of the animals were counted. The values shown represent means and standard deviations of data for three animals in each group. b Soft organs included kidneys, spleen, heart, and lungs. " Intestines were not cleaned. w a s c o l l e c t e d f r o m b o t h g r o u p s a n d , as c a n b e s e e n in T a b l e III, t h e t o t a l a m o u n t o f feces excreted by the cadmium-treated g r o u p w a s s u b s t a n t i a l l y l e s s t h a n t h a t excreted by the control group. There was a corresponding diminution in the amount of 65Zn e l i m i n a t e d . F o o d c o n s u m p t i o n o f t h e C d - t r e a t e d m i c e w a s a b o u t 40% t h a t o f t h e c o n t r o l s d u r i n g t h e 7 - d a y p e r i o d . T h e red u c t i o n in f e c a l 65Zn c o u l d a c c o u n t for o v e r 60% o f t h e t o t a l b o d y i n c r e a s e . T h e r e d u c t i o n i n f e c a l e~Zn e x c r e t i o n w a s Control Liver Intestine Soft organs b Front carcass c Rear Carcass c Weight of 7day fecal excrement (g} Total cpm of 7-day excrement 92,300 • 100,200 • 36,400 • 335,800 + 311,800• 58 7,000 16,000 2,900 17,700 16,000 3,856,000 Cd treated 200,000• 141,000• 52,900• 420,900• 366,300• 30 39,100 40,200 14,800 75,200 82,400 2,800,000 Ten mice were pretabeled with ~Zn (approximately 20 pCi total per animal). Three days after the last injection of 65Zn, five animals were injected with CdCI2 (2.5 mg of Cd/kg) and the other five with isotonic saline. The animals were housed in metabolic cages for the next 6 days, during which time the combined excrement for a group was collected. Seven days after the CdCI2 injection, the mice in both groups were killed. Tissue radioactivity values represent means of data for five mice. Standard deviations are indicated. h Soft organs included kidneys, spleen, heart, and lungs. Carcass division made at bottom of rib cavity. n o t a s p e c i f i c e f f e c t ; r a t h e r , t o t a l f e c a l excretion was inhibited. In a third related experiment with mice, the fecal matter was ashed and analyzed for various metals. It can be seen in Table IV that, whereas the absolute amounts of several metals elimin a t e d w e r e l o w e r in t h e C d - t r e a t e d g r o u p , the concentrations were unaltered. In this t h i r d e x p e r i m e n t , t h e d e c r e a s e i n f e c a l ~Zn ZINC CONTENT OF Cd-INDUCED THIONEIN TABLE IV EFFECT OF Cd ON EXCRETION IN THE MOUSEa Sample ~Zn (cpm/tissue) Liver Soft organs Intestines cleaned Testes Total fecal matter Total excrement (g) Total fecal Zn Control Cd treated 83,500 -e 4,100 42,800 _+ 1 , 2 0 0 95,700 2:22,200 134,300+ 13,000 41,800 + 2,100 105,500_+ 9,700 8,600 _+ 1,300 369,500 7,700 _+ 800 189,200 3.2 1.5 613 289 191 185 122 50 38 32 2800 1400 880 900 (#g) Zn (pg/g of feces) Total fecal Cu (#g) Cu (#g/g of feces) Total fecal Fe (~g) Fe (#g/g feces) of Two groups of three mice prelabeled with ~Zn (approximately 20 ~tCi per animal) were injected with 2.5 mg of Cd/kg or isotonic saline 2 days following the last ~Zn injection. The animals were then housed in metabolic cages for 2 additional days and were killed the following day. The radioactivity data represents mean values _+ standard deviations. The fecal excretion was pooled and counted for each group. in the experimental group was substantially more than the increase observed in the liver. In the mouse, Cd-induced zinc mobilization must result in large part from an effect on intestinal motility. This same effect was observed in experiments with rats maintained in metabolic cages. Cadmium administration caused a reduction in total intestinal excretion rather than a specific decrease in zinc elimination. In two separate metabolic experiments with rats, the reduction in fecal SSZnaccounted for at least 70% of the increased hepatic e~Zn uptake. Cadmium was found to have virtually no effect on urinary excretion of zinc. There was no difference between control and experimental groups in the total amount of urinary zinc excreted in a 24-h period, but analysis of SSZn showed a small but reproducible 471 increase in radioactivity in the urine of Cdtreated animals. Urinary 65Zn excretion in a 24-h period was less than 10% of the total fecal excrement. The urinary pathway is known to be of little importance in normal zinc excretion. The data suggested that an obstruction of intestinal motility would lead to an elevated zinc uptake in liver. To test this hypothesis, the intestines of a group of rats were surgically ligated approximately halfway through the duodenum. Forty-eight hours later, hepatic zinc levels were analyzed and compared to values from shamoperated rats. Livers from the intestine-ligated rats had a Zn content of 37 /~g/g compared to a Zn content of 27.7 #g/g of liver in the sham-operated animals. The absolute amount of zinc also showed a 35% increase. The zinc content in the thionein fraction increased 12-fold over control levels. If Cd-induced zinc mobilization is a result of an inhibition of excretion and an enhancement of dietary absorption, then animals maintained on a Zn-deficient diet should not show the zinc effect. It is known that animals maintained on such a deficient diet show an enhanced absorption capacity for zinc and a decreased rate of loss of endogenous Zn (14). Since the deficient diet contained only 0.5/~g of zinc/g, intestinal absorption could not supply adequate zinc for significant mobilization. A group of rats was maintained for 25 days on a zinc-deficient diet and deionized water, while a second group was given the same diet supplemented with 60 ~g of zinc/g. Deficient rats showed the expected decreased rate of weight gain (average increase of 20 g, compared to 91 g for Zn-supplemented animals). After 25 days, half of the deficient animals were given a single subcutaneous injection of CdCl2, and 24 h later the rats in all three groups were killed. As can be seen in Table V, the zinc contents in tissues of the deficient animals were lower than those in the supplemented rats. Yet after cadmium administration, the zinc level showed the normal enhancement in liver as well as in kidney and in bone. Fractionation of the heat-treated liver supernatant on Sephadex G-75 showed the increased hepatic zinc to WINGE, PREMAKUMAR, AND RAJAGOPALAN 472 TABLE V Zn- deftciem EFFECT OF C d ON Z n CONTENT IN TISSUES OF Z n DEFICIENT RATS a Tissue Liver Kidney Tibia Backbone Zn-suppiemented diet 30.3 • 1.2 21 • 1.5 61.5 + 0.9 64 • 9.7 Zn-deficient diet Zn-deficient diet + CdC12 Zn (~g/g of tissue) 24.8 • 0.6 44.2 • 17.5 • 3.6 20.5 • 32.3 • 7.4 39 • 23 • 5.7 28 • 7.1 1.9 10.8 7.0 Cd (~g/g of tissue) Liver Kidney Tibia Backbone _b --~ -- 52 • 4.7 1 2 + 3.2 -- 3 . 6 +_ 0 . 4 -- 2.5 + 0.2 "Two groups of three rats were maintained on a Zn-deficient diet (0,5 #g of Zn/g) for 25 days, while a third group was fed the diet supplemented with 60 #g of Zn/g. CdCl2 was injected subcutaneously to one group of Zn-deficient animals at a dose of 2.5 mg of Cd/kg body weight, while the remaining two groups received isotonic saline. The animals were killed 24 h following the injections. The results represent mean values • standard deviations. bCd levels in these tissues were near background values. elute with cadmium in the thionein fraction (Fig. 5). T h e identification of this peak as thionein was confirmed by ion-exchange chromatography, where both major polymorphic forms of Cd,Zn thionein were observed. Identical results were obtained when the experiment was repeated. In the zinc-deficiency experiment, alcohol dehydrogenase was assayed in liver extracts as another measure of the degree of deficiency. T h e r e was a 25% decrease in this activity in deficient rats (Table VI). T h e hepatic enzymatic activity in the Cdtreated rats was significantly higher than t h a t in the Zn-deficient animals. This suggested t h a t the mobilized zinc could be utilized for tissue zinc demands in addition to being incorporated into thioneine. T o substantiate this observation, a group of rats was placed on a low-zinc diet (7 ~g of Zn/g of diet) and given 20% ethanol in their drinking water. These dietary conditions have been reported to result in a partial zinc depletion in certain tissues (16). After 3 weeks on this regimen, the animals were 2.0 1.o i ~~~l=edl=14.0 2.0 Zn-dehc, Cd enr+ 2.C - - I . ~ I - 4.0 1,0 ~ ~ ~ L J~" 2.0 ~o 20 30 40Fracti 50 o60nNumber 70 80 90 O0 FIG. 5. Sephadex 0-75 column chromatography of the soluble protein fraction from livers of zinc-deficient rats. Experimental details are described in Table V, footnote a, and column conditions are as described in the legend to Fig. 3. The homogenate was centrifuged finally at 100,000g prior to column application. The soluble protein fraction was not heat treated in either the Cd-treated or the control homogenates. divided into three groups, one group receiving a single subcutaneous injection of CdCl2 (2.5 mg of Cd/kg), the second group a single subcutaneous injection of ZnC12 (5 mg of Zn/kg), and the third group isotonic saline. In both groups receiving a metal injection, not only was the hepatic zinc concentration substantially higher t h a n the control level, but the alcohol dehydrogenase activity was elevated markedly. T h e enzyme activity was not affected by Cd administration to control rats. T h e Zn-deficiency experiments were repeated using mice as the experimental animals. T h e animals were maintained on the Zn-deficient diet for 25 days. T h e dietary conditions were the same as described in Table V. C a d m i u m was administered to two groups and the animals were killed 8 days later. As can be seen in Table VII, Cdtreated mice showed enhanced liver Zn levels in both Zn-deficient and Zn-supplemerited groups. T h e elevation in hepatic ~Zn could largely be accounted for by a decrease in fecal ~Zn. DISCUSSION A variety of metal ions, including Cd, Hg, Ag, Au, and Zn, has the ability to induce thionein formation in rat liver. E a c h of ZINC CONTENT OF Cd-INDUCED THIONEIN 473 TABLE VI EFFECT OF Cd ON ALCOHOL DEHYDROGENASE ACTIVITY IN RAT LIVERa Experiment Zn deficiency Ethanol feeding Group Alcohol dehydrogenase activity (units/g of liver) Zn supplemented Zn deficient Zn deficient + Cd Zn deficient Zn treated Cd treated 6.67 • 4.98 • 5.78 • 7,0 • 9.45 • 9.95 • 0.6 0.3 0.3 0.4 0.2 0.2 Zn (#g/g of liver) 30.3 24.8 44.2 25 33.6 36.6 • • • • • • 1.2 0.6 7.1 1.5 1.5 3.0 Two different experiments were conducted. Aliquots of supernatants, prepared by centrifugation at 100,000g of the homogenates from the animals described in Table V, were dialyzed against 10 mM potassium phosphate, pH 7.8. These samples were assayed for alcohol dehydrogenase activity as described by Bonnichsen and Brink (15). In the second experiment, six rats were placed in plastic cages and were fed a normal-protein test diet (low zinc content) and 20% ethanol in deionized water. After 20 days, two rats were injected subcutaneously with CdCt2 (2.5 mg of Cd/kg), two with ZnC12 (5.0 mg of Zn/kg), and the third two with isotonic saline. The animals were killed 24 h later. Alcohol dehydrogenase was assayed in the supernatant as described above. TABLE VII EFFECT OF Zn DEFICIENCY ON Zn MOBILIZATIONa Sample Total counts per minute per sample Zn supplemented Zn supplemented + Cd Liver Soft organs 4-Day fecal matter Zn deficient Zn deficient + Cd 217,400 + 16,400 93,400 + 6,600 313,900_+ 25,900 108,600_+ 15,300 322,600-e 68,100 142,400• 34,200 614,200+ 63,900 241,400• 37,500 3,400,000 2,775,000 2,293,500 803,000 A Fecal cpm [Def [Suppl A Liver cpm [(Def [(Suppl A Soft organ [(Def cpm [(Suppl + + + + (Def (Suppl Cd) Cd) Cd) Cd) + + - Cd) ] Cd) ] Def ] Suppl] Def ] Suppl] = 1,490,500 = 625,000 = 1,458,000 - 482,600 -- 500,000 = 76,000 "Twenty mice were divided into four groups and maintained on special diets for 3 weeks. At that time, each animal was given 15 #Ci of ~Zn. Four days later, one group of Zn-supplemented and one group of Zn-deficient mice were given one subcutaneous injection of Cd (2.5 mg of Cd/kg). All animals were killed 8 days after the ~SZn injection. The tissue radioactivities listed are mean values, while the fecal counts are total counts per minute of fecal matter per group. The changes in radioactivity listed represent differences in the summation of counts per minute from the five mice in each group. these ions also causes a mobilization of zinc to the liver, where the zinc becomes incorporated into thionein molecules. Other cations, such as Pb, Pd, Sn, and Cu, do not elicit the same response. In this study, cadmium has been shown to induce zinc mobilization in normal rats and mice, partly by enhancing dietary zinc absorption but primarily by interfering with intestinal excretion. Intestinal ligation was also shown to result in zinc mobilization. Presumably, any inhibition of intestinal mobility will produce a similar body retention of zinc. The obstruction of fecal elimination may permit the endogenous zinc from bile, pancreatic juices, and other digestive secretions to be absorbed into the blood. This is also suggested by the studies of Cotzias et al. (17), who showed that after an injection of CdSO4 to rabbits preloaded with ~Zn, there was a transient increase in plasma radioactivity. Once this absorption into blood occurs, the enhanced zinc levels are known to be rapidly cleared by accumulation in the soft tissues (18). In rats and mice, the liver and pancreas are two tissues that initially absorb the greatest amounts of blood zinc (19). The observation that hepatic thi- 474 WINGE, PREMAKUMAR,AND RAJAGOPALAN onein has a greater zinc content than renal thionein is presumably a result of the ability of the liver to accumulate or clear plasma zinc to a greater extent than the kidney. But in animals where plasma zinc is cleared to the same extent by liver and kidney, e.g., dog {20), the hepatic and renal thioneins might contain the same zinc content after Cd treatment. It is well known that the amount of fecal excrement is dependent on the amount of diet consumed. Restriction of food intake should therefore reduce fecal excretion. Bremner and Davies (21) reported that restriction of food intake in the rat led to a greater zinc content in the thionein fraction and that the greater the degree of starvation, the greater the increase in the thionein zinc level. Cotzias and Papavasiliou (22) reported that injected cadmium perturbed the distribution of zinc in mice such that liver zinc increased while gastrointestinal zinc decreased. They also observed that the effects of injected and oral cadmium were somewhat different. The Cd-induced zinc effect was the subject of a communication by Gunn et al. (23). They interpreted a SSZn metabolic experiment as showing that cadmium inhibited fecal excretion of Znss. The gastrointestinal effect of cadmium is not specific for zinc; rather, it is a result of an inhibition of the fecal excretion. The cadmium effect may be more complicated than merely affecting intestinal movement. Zinc has been implicated to have a functional role in taste and appetite {24), and recently Henkin et al. (25) isolated a zinc metalloprotein from human parotid saliva. If cadmium antagonized the zinc function in appetite, the result may be analogous to starvation-induced zinc mobilization. Another aspect probably contributing to the overall observation of Zn mobilization is that thionein once induced may sequester zinc in tissues, especially the liver. This increased liver retention of zinc would result in an elevation of the hepatic Zn content and therefore a concomitant rise in thionein Zn. In a recent study of the turnover of rat liver Cd,Zn thionein, Chen et al. (26) reported that both the zinc and the radioactive protein label turned over with a half-life of 4.2 days, whereas the turnover of cadmium was substantially longer. The continued turnover and reinduction of thionein in liver resulting from an inability of the tissue to eliminate cadmium may lead to a hepatic Zn trap. The fiver would absorb plasma zinc, which would then be complexed to thionein. The extent of this trapping would be dependent on the hepatic Cd concentration. This mechanism could explain the observation that thionein isolated 6 months after a single dose of cadmium showed a normal Cd/Zn ratio. It is not known whether the eight metal-binding sites on thionein are identical, but it'is conceivable that two sites have a high specificity for zinc. It would be informative to isolate bulk quantities of Cd-induced ttlionein 6 h after the Cd injection to determine the stoichiometry of Cd binding. Such molecules were shown to be devoid of zinc. It is not known whether mercury, silver, and gold salts induce zinc mobilization through an effect on the excretory pathway or dietary absorption, but certainly Zn thionein could be formed by a thionein retention mechanism mentioned above. Zinc deficiency does not impair the ability of rats or mice to form metallothioneincontaining zinc. It is apparent that, in Zndeficient animals, the predominant process involved is a decreased excretion of endogenous Zn resulting from the trapping effect of thionein. The data in Table VII indeed show that the Cd-induced decrease in fecal Zn excretion is much greater in the Zndeficient animals than in the controls. It is intriguing that, despite the various body needs for zinc in deficient animals, a substantial quantity of the metal is mobilized to the liver, where it is incorporated into thionein. Some of the mobilized zinc can be utilized for other purposes, as demonstrated by alcohol dehydrogenase--an enzyme whose activity has been demonstrated to correlate with the hepatic zinc content (27). Two groups have demonstrated that humans accumulate cadmium and zinc in the renal cortex with age (28, 29). Human renal cortex is known to contain Cd,Zn thionein (9), so it is conceivable that the continuous turnover and reinduction of thionein in the ZINC CONTENT OF Cd-INDUCED THIONEIN cortex resulting from an inability to elimin a t e C d m a y l e a d to s e q u e s t e r i n g o f zinc. The concentration of hepatic thionein r e s p o n d s t o s e v e r a l m e t a l ions, b u t i t a p p e a r s t h a t p h y s i o l o g i c a l l y its r e s p o n s e is d i r e c t e d t o w a r d zinc. C i r c u m s t a n c e s l e a d ing t o a n a b n o r m a l l y h i g h p l a s m a zinc level, such as excessive Zn consumption, starvation, o r i n t e s t i n a l o b s t r u c t i o n , m a y r e s u l t in substantial Zn thionein formation. Thio n e i n m a y a c t t o r e g u l a t e t h e free t i s s u e c o n c e n t r a t i o n o f Zn, t h e r e b y p r e v e n t i n g a n y c y t o t o x i c e f f e c t s o f t h e free ion. F u r t h e r roles such as Zn storage or intracellular transport may be of importance. The apparent ability of thionein to detoxify heavy metal ions may be only a fortuitous consequence of the physiochemical similarities of t h e s e i o n s a n d zinc. 475 11. KRATKY, O., LEOPOLD, H., AND STABINGER, H. (1973) Methods Enzymol. 27, 98-110. 12. NORDBERG, G. F., PISCATOR, M., AND LIND, B. (1971) Acta Pharmacol. Toxicol. 29, 456-470. 13. GILBERT, I. G. F., AND TAYLOR, D. M. (1956) Biochim. Biophys. Acta 21, 545-551. 14. MILLER, W. J., MARTIN, Y. G., GENTRY, R. P., AND BLACKMAN, D. M. (1968) J. Nutr. 94, 391-401. 15. BONNICHSEN, R. K., AND BRINK, N. G. (1955) Methods Enzymol. 1, 495-500. 16. WANG, Z., AND PIERSON, R. N. (1975) J. Lab. Clin. Med. 85, 50-58. 17. COTZIAS, G. C., BORG, D. C., AND SELLECK, B. (1961) Amer. J. Physiol. 201, 63-66. 18. OKUNEWICK,J. P., POND, B., AND HENNESSY, T. G. (1962) Amer. J. Physiol. 202, 926-930. 19. RUBINI, M. E., MONTALVO,G., LOCKHART, C. P., AND JOHNSON, C. R. (1961) Amer. J. Physiol. 200, 1345-1348. 20. SHELINE, G. E., CHAIKOFF, I. L., JONES, H. B., AND MONTGOMERY,M. L. (1943) J. Biol. Chem. REFERENCES 1. MARGOSHES, M., AND VALLEE, B. L. (1957) J. Amer. Chem. Soc. 79, 4813-4814. 2. WINGE, D. R., PREMAKUMAR, R., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 170, 242-252. 3. KAGI, J. H. R., HIMMELHOCH,S. R., WANGER,P. D., BETHUNE, J. L., AND VALLEE, B. L. (1974) J. Biol. Chem. 249, 3537-3542. 4. NORDBERG,G. F., NORDBERG, M., PISCATOR,M., AND VESTERBERG, O. (1972) Biochem. J. 126, 491-498. 5. WESER, U., RuPP, H., DONAY,F., LINNEMANN,F., VOELTER, W., VOETSCH, W., AND JUNG, G. (1973) Eur. J. Biochem. 39, 127-140. 6. KIMURA, M., OTAKI, N., YOSHIKI, S., SUZUKI, M., HORIUCHI, N., AND SUDA,T. (1974) Arch. BIOchem. Biophys. 165, 340-348. 7. RUGSTAD, H. E., AND NORSETH, T. (1975) Nature (London) 257, 136-137. 8. NOEL-LAMBOT,F. (1976) Experientia 32, 324-325. 9. PULIDA, P., KAGI, J. H. R., AND VALLEE, B. L. (1966) Biochemistry 5, 1768-1777. 10. KOJIMA, Y., BERGER, C., VALLEE, B. L., AND KhaI, J. H. R. (1976) Proc. Nat. Acad. Sci. USA 73, 3413-3417. 147, 409-414. 21. BREMNER, I., AND DAVIES,N. T. (I975) Biochem. J. 149, 733-738. 22. COTZIAS, G. C., AND PAPAVASILIOU,P. S. (1964) Amer. J. Physiol. 206, 787-792. 23. GUNN, S. A., GOULD, T. C., AND ANDERSON, W. A. D. (1962) Proc. Soc. Exp. Biol. Med. 111, 561-562. 24. HENKIN, R. I., SCHACHTER, P. J., RAFF, M. S., BRONZERT, D. A., AND FRIEDEWALD, W. T. (1975) in Clinical Applications of Zinc Metabo- lism (Pories, W. J., Strain, W. H., Hau, J. M., and Woosley, R. L., eds.), pp. 204-207, Charles C. Thomas, Springfield, Ill. 25. HENKIN, R. I., LIPPOLDT, R. E., BILSTAD, J., AND EDELHOCH, H. (1975) Proc. Nat. Acad. Sci. USA 72, 488-492. 26. CHEN, R. W., WHANGER,P. D., AND WESEIG, P. H. (1975) Biochem. Med. 12, 95-105. 27. REINHOLD, J. G., PASCOE, E., ARSLANIAN, U., AND BITAR, K. (1970) Biochim. Biophys. Acta 215, 430-437. 28. SCHROEDER, H. A., NASON, A. P., TIPTON, I. H., AND BALASSA,J. J. (1967) J. Chron. Dis. 20, 179-210. 29. PISCATOR, M., AND LIND, B. (1972} Arch. Environ. Health 24, 426-431.