Distribution of metallothionein-bound cadmium and cadmium chloride in mice: preliminary studies.

Metallothionein from livers of mice was isolated by gel chromatography and isoelectric focusing. One of two forms thus obtained contained 32 percent cysteine. This form, labeled in vitro with 109Cd, was injected intravenously in mice, and the distribution of 109Cd was studied. Animals killed after 4 hrs had over 80 percent of the injected dose in the kidneys. Protein obtained after gel chromatography, containing both forms of cadmium-binding protein, was also labeled in vitro with 109Cd and injected intravenously. Animals killed 4 hrs after injection had 50 percent of the injected dose in the kidneys. Whole-body measurements and wholebody autoradiography demonstrated that approximately 40-60 percent of the injected dose had been excreted in urine. The results show a selective accumulation of metallothionein-bound cadmium in the kidney and indicate possible differences in distribution and excretion of cadmium depending on binding to different forms of low molecular weight cadmium-binding proteins. ImagesFIGURE 3.FIGURE 4.


Introduction
A low molecular weight protein with an unusually high content of cysteine was first reported by Margoshes and Vallee (1). Kagi and Vallee (2) published further data on this protein, at which time they gave it the name, metallothionein. A subsequent finding was that cadmium induced the synthesis of this protein (3).
The metabolic role of metallothionein is still under discussion (4), one suggestion being that it acts as a detoxifying agent for cadmium (5). In animal experiments, this role has been appeared to involve on the one hand a protective effect against testicular damage (6) and on the other hand a damaging action upon the kidney (7). A  been advanced implying that metallothionein plays a part in the redistribution of cadmium from the liver to the kidney which occurs in long-term exposure to cadmium (5).
The studies presented here were designed to elucidate the fate of 109Cd-labeled metallothionein when given intravenously. The study was performed at two laboratories and therefore somewhat different techniques as well as two different mouse strains were used.

Labeling of Metallothionein
Mouse liver metallothionein was prepared as described by Nordberg,Nordberg,and Piscator (8). The protein, obtained after ultracentrifugation and chromatography on G-75 and G-50 Sephadex but not further purified, was designated cadmiumbinding protein (Cd-BP).

December 1975
Characterization of this Cd-BP by isoelectric focusing revealed two main components. One contained 61% of the total cadmium and very little zinc, and had a pI of approximately 4 (identical with the cadmium-metallothionein form 1 to be described below). Another contained 24% of the total cadmium and the main part of the zinc that was present in the preparation. This component had a pl of approximately 6.
Isoelectric focusing was also employed for further purification of metallothionein. In order to optimize the output of purified protein, fractions corresponding to a pH-interval of 3.9-4.2 were pooled and deampholytized. The protein thus obtained was designated metallothionein form 1 (Cd-MT1). Characterization by amino acid analysis gave only minor differences from earlier findings by Nordberg,Nordberg,and,Piscator (8). A high purity of the present metallothionein (Cd-MT1) was indicated by the high content of cysteine (32 residues -% ) and the absence of aromatic amino acids. The metal content of lyophilized Cd-MT1 was 10% Cd (w/w) and approximately 0.4% Zn (w/w). The E250/E28o ratio was 17. The labeling of Cd-BP and Cd-MT1 was performed in the same way. The respective proteins were exposed to l09Cd in vitro in an aqueous medium containing Tris-NaCl (Tris buffer 0.01M in 0.05M NaCl, pH 8.0, for the Cd-BP; Tris buffer p.AM in 0.5M NaCl, pH 8.0, for Cd-MT1). That all l09Cd had been bound to the protein and that virtually no free cadmium remained were checked by chromatography on G-50 and G-75 Sephadex (Figs. 1 and 2O. The radioactive cadmium peak appeared at the same elution volume as has the nonradioactive metallothionein cadmium peak earlier.

Animals
Twenty-five male C57BL16JH (J67) mice were obtained from the Jackson Laboratory, Bar Harbor, Maine 04609, U.S.A. The mice, which had body weights of 25-33 g, were divided into two groups for studies on 109Cd-MT1 and 1O9CdCl2 in comparable doses (Table 1).
Twenty male CBA-mice from the Institute of Genetics, University of Stockholm, Sweden, with body weights of 20.0-25.3 g, were divided into two groups and used for studies on 109Cd-BP and parallel injections of 1O9CdCl2 ( Table 2).

Treatment of Animals
All animals were given a single intravenous injection in a tail vein. Fourteen C57BL mice were given l19Cd-MT1, and eleven such mice were given the same doses of cadmium as radiolabeled CdCl2. Subdivision of animals in two dose groups (0.03 and 0.08 mg Cd/kg) is seen in Table 1. In order to collect urine from mice with 4 hr survival time the animals were put in metabolism cages during the interval between injection and killing. Animals were killed after 5 min or 4 hr by heart puncture and cervical dislocation. Organs were removed and weights recorded. Cadmium content was calculated from values obtained by gamma counting of the organs (6).  Ten CBA-mice were given Cd-BP and ten mice were given radioactive cadmium as CdCl2. Each animal was given approximately 1.6 ,tg Cd and 0.4 jig Zn, corresponding to a dose of 0.075 mg Cd/kg body weight and 0.02 mg Zn/kg body weight. The cadmium/zinc ratio is the same as in the Cd-BP. One or two animals were killed after 1, 5, and 20 min and 24 and 96 hr ( Table 2). After injection and just before killing all animals were subjected to whole-body counting.

Other Methods
Autoradiography was performed on the animals treated with 109Cd-BP and the corresponding ones injected with 109CdCl2. The method was described by Ullberg (11 ). In the main, sections of the frozen mouse were prepared at 20 j,m on a tape and apposed to Structurix or Kodirex X-ray film.
Scintillation counting of organs removed from the thawed corpses of the mice (after sectioning) was performed in a gamma counter as described by Nordberg (6).

Results and Discussion
Scintillation Counting Table 1 gives the amounts of cadmium found in liver and kidney after injection of 109Cd-MT1. After 5 min, 38.6% (mean) of the total injected dose has already accumulated in the kidney in the lower dose group. The corresponding figure for the higher dose group was almost the same (38.2% ). By 4 hr after the injection, the animals given the lower dose had an average of 95.4% of the total injected dose in the kidneys, and the corresponding figure for the higher dose was 81%.
In some cases more than 100% of the injected dose were recovered in liver and kidney, particularly in the lower dose group. Indeed, an error of the order 20% may easily arise and be explained by the small volume of solution injected (0.05 ml), since the syringe was graduated in 0.01 ml units. In the higher dose group, larger volumes (0.15 ml) were used, the relative error upon injection therefore being smaller.
It is further evident from Table 1 that more than 10 times as much cadmium was accumulated in the kidneys when the cadmium was injected as Cd-MT1 than as CdCl2; liver values, on the other hand, were lower.
Values for animals injected with Cd-BP and with survival times corresponding to those in the Cd-MTI study are also given in Table 1. In these animals approximately 10-fold larger amounts of cadmium were found in kidneys than with the corresponding CdCl2 animals.
A direct comparison of Cd-BP animals with CdMT1 ones is obviously somewhat limited because different strains of mice were used for the two studies. It might be appropriate to point out, however, that the renal accumulation expressed as per cent of injected dose in Cd-MT1 animals (81% ) seems higher than when approximately the same dose of cadmium was injected as Cd-BP (50% ).
This observation was further supported by the whole body measurements in Table 2. Whereas a very minor tendency, i.e., approximately 10%, towards a decrease arose in CdCl2 animals during the 96 hr, the whole body values of the Cd-BP mice decreased by approximately 40-60% by 4-96 hr after injection.
Urine values for the CdMT1 injected mice (4-hr survival time) showed a broad range for the lower dose group. They varied from 0.03-1.23% of the injected dose. In the higher dose group, corresponding values were 0.03-2.18% of the injected dose. For the Cd-BP injected mice some occasional excretion values were also obtained. One 4-hr mouse excreted 0.4 ,ug (24% ) of cadmium in urine and feces. The second 4-hr mouse excreted less than 0.0001 jig Cd in feces during the same time interval. Urine was not collected. From this it seems most likely that the cadmium has been excreted via urine. This also indicates a difference in Cd-excretion of the two protein forms Cd-MT1 and Cd-BP. The mice injected with 1O9CdCl2 had a low excretion of cadmium in the urine and the values (4 hr) ranged from 0.02 to 0.08% of the injected dose. Autoradiography Distribution among Organs: The general distribution pattern of injected l09CdCla was the same as that reported by Berlin and Ullberg (9) and Nordberg and Nishiyama (10) with the highest concentration in the liver and somewhat lower concentrations in pancreas and kidney at the survival times employed (2 min-1 day).
A markedly ditterent pattern mace itsent eviaent in autoradiograms from animals injected with 109Cd-BP. By 2 min after injection, substantial uptake of cadmium in the renal tissue had already occurred, this organ standing out with considerable blackening against a very pale background representing other tissues. Blackening of an intensity similar to the one corresponding to the kidney was also seen to represent the contents of the urinary bladder in 20-min animals, but not in animals having shorter or longer survival times. The difference in the distribution patterns between CdCl2 mice and Cd-BP mice is illustrated in Figure 3.
Distribution within the Kidneys: Autoradiographic studies employing the whole body sectioning technique also enable determination of distribution among various structures within organs (9,11 ). In the present study the distribution within the kidney is of particular interest. In autoradiograms from animals injected with 109CdCl2, the distribution pattern in the kidney was in accord with that reported previously (9, 10, 12), i.e., a maximum spotwise darkening in the outer zone of the kidney cortex, probably corresponding to the most proximal tubule. At variance with what was reported by Berlin and Ullberg (9), the present Environmental Health Perspectives autoradiograms obtained at the shortest survival times represents an excretion of cadmium into the urine. Autoradiograms from longer times after injection showed most of the cadmium retained in these 109Cd-BP-injected mice to be confined to the kidney cortex (Fig. 4). The spotwise appearance of the blackening in the outer cortex of these animals is similar to the pattern unfolded (12) for cadmium chloride injected mice and shown to correspond to an uptake in the first part of the proximal tubule. More detailed studies are warranted in order to define more precisely the localization of metallothionein-bound cadmium. autoradiograms demonstrated that the darkening in the renal cortex, even at the darkest spots, never exceeded the darkening of the liver during the shorter times after injection. This may be explained by the highest doses of cadmium used in this study, since it is known (5) that a smaller proportion of cadmium will be found in the kidney of experimental animals given larger doses. The whole body countings (Table 2) show that the amount of cadmium retained in the group injected with 1O9CdCl2 was approximately constant, while appreciable excretion of cadmium occurred already between 20 min and 4 hr in mice injected with 109Cd-BP. This suggests that the blackening in the central parts of the kidney observed in In summary, the present results show that, once it has reached the blood, metallothionein-bound cadmium is transported to the kidney where it accumulates selectively. This supports our previously advanced theory pointing to an involvement of metallothionein in the redistribution of cadmium from liver to kidney in long-term exposure and a role of this protein in the elicitation of renal tubular effects of cadmium. Acknowledgement A grant from the Swedish Medical Research Council (Project 26X-775) is gratefully acknowledged.
The skillful technical assistance of Mrs. Maud Pannone is gratefully acknowledged.