Metal-induced formation of metallothionein in rat liver

Metallothionein

was first isolated as a cadmium-containing protein from equine renal cortex by Margoshes and Vallee in 1957 (1). Subsequent studies with this cytoplasmic protein from a variety of sources including human kidney (21, equine kidney and liver (3,4), rabbit liver (51, chicken liver (61, and rat liver (6, 7) revealed an amino acid composition marked by an unusually high cysteine content, ranging from 26-33 mol%, and absence of aromatic amino acids. Metallothioneins from various sources have been shown to contain cadmium, zinc and traces of mercury and copper (l-7).

In rabbits (51, chickens (61, and rats (6)(7)(8)(9), accumulation of large amounts of metallothionein in the liver is accomplished by injection of cadmium. Other reports have indicated that copper (10, 111, zinc (8, 121, and mercury (13 -15) also induce the formation of low molecular weight soluble proteins. These proteins have usually been assumed to be metallothioneins, although no definitive proof has been advanced to that effect. The protein induced in rat liver by copper has been established to be distinct from metallothionein and has been termed copper-chelatin (16,17). In view of the possible multiplicity of related proteins, it was of interest to characterize the proteins induced by zinc and mercury in rat liver and to determine their relationship to metallothionein.

In this paper we present evidence for the identity of the proteins induced in rat liver by zinc, mercury and silver with cadmiuminduced metallothionein.

RESULTS

Animals in groups of three were injected subcutaneously with cadmium chloride, mercuric chloride, zinc chloride or silver nitrate. The pooled livers from each group were rinsed and homogenized with five volumes of 0.01 M potassium phosphate, pH 7.8. The homogenates were centrifuged at 27,000g for 10 min, and the supernatant fluids therefrom were recentrifuged at 100,OOOg for 60 min to obtain particle-free preparations.

The Figure 1 shows the elution profiles of liver extracts obtained from rats injected with cadmium.

In the mercury, silver and zinc groups, analogous low molecular weight fractions (tubes 90-100) containing the corresponding metals were observed. In the cadmium, mercury and silver groups, zinc was also present in these fractions.

Similar chromatography of the soluble fraction from control rats revealed the absence of detectable cadmium and silver in this region but showed a minor zinc peak.

Fractions in analogous regions in the elution profiles (tubes 88-102) were pooled and concentrated by lyophilization.

Following resuspension and dilution with deionized water to a conductivity below 5 mmho/cm, the samples were subjected to acetone fractionation in three stages: O-40, 40-60, 60-80% (v/v). The metalloproteins were found to fractionate predominantly in the 60-80% acetone fractions. A marked increase in the total protein content of this fraction obtained from each of the four experimental groups, compared to the control group, was a striking feature of this experiment (Table I). The elevated zinc content of all of the metalloprotein fractions is also evident.

The 60-80% acetone fractions were dialyzed against 0.01 M Tris-Cl, pH 8.6, at 25°C. Each sample was then chromatographed on a DEAE-cellulose3 column (4 x 1.9 cm) equilibrated at 4°C with the same buffer. Figure 2 shows the typical elution for the Cd-, Hg-, Ag-and Zn-proteins.

In each case, two major metal-containing fractions coinciding with protein peaks were eluted in volumes corresponding to conductivities of about 0.3 and 0.6 mmho/cm. For convenience, the two elution peaks are referred to as A (0.3 mmho fraction) and B (0.6 mmho fraction).

The two silver-containing fractions invariably displayed an excess of zinc over silver.

To investigate the apparent identity of elution behavior of each of the two frac-tions in all groups, the A and B forms of the cadmium protein were mixed with the corresponding forms of the mercury protein, and the resulting mixtures chromatographed on DEAE-cellulose under conditions identical to those described earlier.

In each case mercury and cadmium eluted from the column in congruence.

The two forms of each metalloprotein were dialyzed against 5 mM potassium phosphate, pH 7.8, concentrated by lyophilization, and subjected to electrophresis in 7.5% polyacrylamide gels. Figure 3 shows the Coomassie blue-stained protein bands in each case. The A forms of all of the metalloproteins exhibited bands of identical mobility with Rfof 0.4, while form B in each case revealed a band with Rf of 0.6. Fig. 3 actually were the two forms of the metalloproteins, gels of 203Hg-labeled protein partially purified through the acetone step were sliced into 12 segments of 0.5 cm, and each segment was digested with 0.2 ml of 30% H202 for 16 h at 60°C in sealed vials. Scintillation cocktail was added, and after cooling to 4°C the samples were counted. As can be seen in Fig. 4 as determined by ultraviolet-absorbance optics monitoring at 260 nm, versus square of the distance from the center of rotation (Fig. 51, linearity was observed at each of the two speeds, indicative of a homogeneous pro-tein preparation. From the slope of the lines and a partial specific volume of 0.69 calculated from the amino acid composition according to the method of Cohn and Edsall (201, a weight average molecular weight of 10,200 k 400 was calculated.

To confirm that the two bands visible in

Amino acid analysis was performed on components A and B of the Cd-thionein and the B forms of the mercury and zinc proteins. The composition of the B components of the Cd-thionein and Hg-and Znproteins were exceedingly similar while significant differences were found between the A and B forms of Cd-thionein (Table II). Both forms A and B of Cd-thionein exhibited the unusually high cysteine content characteristic of all metallothioneins, indicating that forms A and B are two variants of metallothionein. This polymorphism had previously been noted in the metallothioneins from equine renal cortex and liver (4), rabbit liver (51, and rat liver (21). Since the mercury, zinc and silver proteins were revealed to be identical to the two forms of Cd-thionein, these results have shown that each of these metals induces the formation of the same two forms of thionein. Calculation of the minimum molecular weight from the amino acid composition yielded a value of 10,100 which correlates well with the value determined by sedimentation equilibrium. The ultraviolet absorption spectra of the Cd-, Hg-, Ag-and Zn-thioneins4 are shown in Fig. 6. The A and B forms of thioneins containing the same metal exhibited similar spectra, but thioneins containing different metals showed nonidentical spectra. This variance is not unexpected, since the absorbance of the Cd-thionein was shown to result from metal-cysteine chargetransfer transitions (22) and different metal-cysteine complexes have transitions corresponding to varying frequencies. In the case of the Cd-thionein, which contained some zinc as well, the molar extinction coefficient of the protein at 250 nm was 79 mM-' cm-lsand the A250nm/ A 280nm ratio was 18.2. The spectrum of the Zn-thionein could be altered to one resem-bling that of Cd-thionein by addition of graded amounts of cadmium ( Fig. 7). At a molar ratio of 2:l of cadmium to zinc, the spectrum was indistinguishable from that of native Cd-thionein, and no further changes were observed when additional cadmium was added to the incubation mixture. The metal content of the two forms of Cd-thionein was found to be five atoms of Cd, two atoms of Zn and 0.1 atom of Cu per molecule of protein. In Zn-thionein, five atoms of zinc were present per molecule with only negligible traces of cadmium and copper. These values were obtained by relating the metal content of metallothionein to the protein content determined by quantitative amino acid analysis of the purified protein. The latter procedure yielded a protein content of 50% relative to the value obtained by the Lowry procedure (23) with bovine serum albumin as standard. Hepatic Zinc Accumulation As described earlier, injection of cadmium, mercury or silver to rats resulted in the elevation of hepatic zinc levels and the presence of zinc in the induced metallothioneins. In an attempt to determine the source of the hepatic elevation in zinc, ani- mals injected twice with cadmium chloride were killed and various tissues and blood samples were collected and metal analysis was performed on homogenates derived from them. As can be seen in Table III, the hepatic zinc content was elevated concomitantly with the cadmium content in cadmium-injected animals. Whereas no significant differences were detected in the zinc contents of other tissues, there was a pronounced decrease in the zinc content of blood in experimental animals. Thus, it appears that the zinc which is mobilized to the liver as a result of cadmium administration arises at least in part from a depletion in the blood content of zinc. The possibility of increased uptake of dietary zinc in cadmium-treated animals cannot be excluded.

Nature of Accumulation of Metallothionein

Three groups of rats subjected to single subcutaneous injections of cadmium chloride, silver nitrate or saline were injected subcutaneously with 50 /..&i each of 13H]lysine and [35S]cysteine. Two hours later, the animals were killed and liver metallothionein was purified as described earlier to obtain the acetone-fractionated sample. There was a substantial increase in labeled proteins containing both isotopes at that stage in both the silver-and the cadmium-treated groups compared to controls. The 35S/3H ratio of the partially purified fraction was 0.6 in the control fraction and 1.2 in both the silver-and the cadmium-treated groups. Figure 8 shows a radioactivity profile from gel slices after electrophoresis of the acetone-fractionated Cd-thionein. Both thionein bands revealed the marked increase in labeling, especially by [35Slcysteine. It sould be noted that elec-trophoresis of the control protein sample after acetone fractionation did not reveal either the presence of metallothionein or the increased labeling of the corresponding gel segments.

Isolation of Metallothionein from Human Liver

Two post-mortem human livers were processed by the same procedure as employed for rat liver. After elution from Sephadex G-75, zinc was the predominant metal detected in the fraction corresponding to metallothionein, with only trace quantities of copper and cadmium present. Ion-exchange chromatography of the acetone-fractionated material on DEAE-cellu- The spectra observed at cadmium/zinc molar concentration ratios of 0 (--), 0.5 (---1, 1.0 c-----l, and 2.0 c--.--l are shown.

lose revealed the presence of the same two forms as in rat liver (Fig. 9). The two forms of the human Zn-thionein resembled the rat liver Zn-thionein in their ultraviolet absorption spectra and in the apparent conversion to a Cd-thionein upon titration with cadmium. Both forms A and B contained 4.6% zinc by weight and less than 0.1% copper.

TABLE II

TABLE III