Results Cd Content and Egress from Cells with and without MT

Cultured cells have been made resistant to otherwise lethal concentrations of the toxic Cd ion, probably by induction of metallothionein (MT) synthesis and binding of Cd to the MT. One human epithelial cell line (HE) and two enzyme-deficient mutants of mouse fibroblasts (Lcells) (Cl 1D and A9) and their Cd-resistant substrains with a high content of MT, have been used to study cellular Cd uptake and metabolism. For cell survival of "wild type" cells, the critical level of intracellular Cd is determined to be around 6 nmole Cd/mg cell protein. Resistant cells can tolerate Cd levels several times above this concentration, if the major part of Cd is bound to MT. The technique of perturbed angular correlation spectroscopy (PAC) has been applied to living Cd-resistant cells. It was shown that > 66% of Cd in the resistant strains was bound to MT, and that MT is apparently freely suspended in the cell cytoplasm. Chelating agents differ in toxicity and Cd-releasing effect on the cells, but apparently remove the non-MT-bound Cd pool. After various periods of Cd omission, either in vitro or in vivo, growing the cells as tumors in athymic nude mice, the stability of Cd resistance in these cells seems to be dependent on the capacity of cells for de novo synthesis ofMT shortly after re-exposure to the metal.


Introduction
Cultured cells can be adapted to high concentrations of the toxic cadmium ion, apparently by induction of synthesis of the Cd-binding protein metallothionein (MT) (1,2). This is one of several indications that MT probably plays a role in Cd detoxification (3). We have recently obtained results which indicate that MT may protect cells from toxic effects of other metals (4) and metalcontaining drugs (5), as well as certain alkylating agents (6) and ionizing radiation (7). In view of the various possible biological functions of MT, we also have found it of interest, in our laboratory, to evaluate the importance of MT as a protective factor against cadmium toxicity. It is the purpose of this paper to present these studies, which are performed mainly as in vitro studies on cultured cells.
Cd Resistance and MT Levels in Cultured Cells Rugstad  epithelial cell line (1) and two enzyme-deficient mutants of mouse fibroblasts (b-cells) (2). The human cells were derived from normal skin, and their epithelial origin was confirmed by phase contrast microscopy and electron microscopy (8). Their human origin was demonstrated by isoenzyme studies and by typing surface antigens by a microcytotoxicity assay (8). The murine cells were two different fibroblast lines: one is designated clone 1D and is deficient in thymidine kinase; the other is designated A9 and is deficient in hypoxanthine-guanine phosphoribosyl transferase. Substrains resistant to otherwise letal doses of CdCl2, namely 100 gmole/L of culture medium, were developed from all three parent cell lines. This was performed by exposing cells to stepwise increased concentrations of CdCl2. The lysates of the Cd-resistant cells, but not those from nonresistant cells, contained large amounts of low molecular weight cadmium binding proteins. These proteins were determined to be metallothionein (MT) by amino acid analysis (5).
There is good evidence that the resistance in these cells is due to induced synthesis of MT and binding of Cd to MT, thereby protecting the cells from the otherwise lethal effect of such high total intracellular concentrations of cadmium.
We also suggest that the pattern of develop-ment of Cd resistance in these cells might be compatible with an amplification of the MT gene. These cells have been used for further studies on Cd metabolism and experiments to reveal functions of the MT. Culture methods and experimental conditions are detailed elsewhere (9)(10)(11)(12).

Results
Cd Content and Egress from Cells with and without MT One of the most common mechanisms of resistance to drugs or other toxic substances is the cells' ability to reduce the intracellular concentration of the substance concerned. This may be caused either by reduced uptake or enhanced efflux from cells. Bakka and Rugstad (9) have investigated the concentration of Cd in the three Cd-resistant cell strains, compared to their parent cell lines by exposing cells to [109Cd]-CdCl2, to reveal whether or not decreased amounts of intracellular Cd might contribute to the Cd resistance in these cells. The results were principally the same for all three Cd-resistant substrains used. Figure 1 shows the results from experiments with the concentration of cellular Cd much higher than that which after about 8 hr incubation injured the nonresistant cells to a degree not compatible with further growth. The critical level of intracellular Cd which cannot be exceeded without lethal consequences on nonresistant cells seems to be 6-8 nmole/mg cell protein, whereas the resistant cells contained 20-30 nmole/mg after 24 hr growth with CdCl2. At that point the resistant cells still had a normal appearance as judged by phase contrast microscopy and were capable of apparently normal growth. After omission of Cd from the medium, the egress of Cd during the first 2 hr occurred more rapidly and to a greater extent from the nonresistant strain than from the resistant strain. After about 2 hr, the Cd egress was the same from both cell strains.
Recent experiments in which the Cd-resistant MT-containing substrain of human epithelial cells was exposed to gold (III) chloride show that these cells in addition are resistant to concentrations of gold chloride which are lethal to the parent cell line, and that the Cd-resistant cell strain is capable of growing with a higher concentration of intracellular gold than that which kills the nonresistant cell strain (4).

Cd-Depleting Effect of Chelating Agents
One of the main problems in treatment of Cd poisoning in humans is the relative inefficiently chelation therapy. One probable reason is that most of the Cd is tightly bound to intracellular proteins, in particular to MT. An efficient chelating agent has to meet several requirements: the chelating agent must have acceptable toxic effects on cells; the complex of the chelating agent and the metal must have acceptable toxic effects; the chelating agent must penetrate into the cells; the complex of the chelating agent and the metal must be released from cells; and the complex must be excreted from the body. Cultured cells with a high content of Cd and MT are considered to be a useful in vitro model for primary screening of different chelating agents regarding some of these requirements, especially those concerning potentially toxic effects of the substances themselves and their ability to increase the release of Cd from the cells. The term "release" is used to designate the overall transfer of metal from cells to the growth medium. Bakka et al. (10) studied the effect of six different metal chelating compounds on cell growth of the Cd-resistant, MTcontaining substrain of the human epithelial cell line and also the effect of chelators on the efflux of Cd from the cells. The release of 109Cd from cells under influence of some of the chelating agents is shown in Figure 2. Of all the substances tested, 2,3-dimercaptopropane-1-sulfonate (BAL-sulph.) caused the most rapid release of Cd from cells during the initial 2 hr incubation, whereas 2,3dimercapto-l-propanol (BAL) and mercaptosuccinic acid (MSA) were the most potent Cd-releasers during the first 24 hr of exposure. The toxicity studies determining cell growth during the influ-  I 1j ence of the chelating agents revealed that BAL was more toxic to the cells than the other agents, indicating that the egress of Cd from cells caused by that agent to some degree probably was a leakage of Cd from cells with injured plasma membrane.

Stability of Cd Resistance and MT Levels
The possible biological function of MT as a detoxifying substance for Cd called for investigations on stability of the Cd resistance and the MT levels in Cd-adapted cultured cells, both of human and murine origin. Endresen et al. (11) expressed the degree of stability of Cd resistance as variations in cell growth during re-exposure to Cd following a period of omission of Cd from the culture medium (Fig. 3). After a 3-week omission period, the resistance was the same in the murine cells, but decreased in the human cell strain to 50% of initial values. Elution profiles obtained after gel filtration of cytosols (Fig. 4) revealed that after 3 weeks there was no detectable MT left in any of the two strains. Both strains had the capacity of rapid de novo MT synthesis even after 5 weeks without Cd. This capacity was best preserved in the mouse fibroblast strain, in which 80% of the previous levels of MT (during continuous growth in Cd-containing medium) could be demonstrated after 24 hr re-exposure to maximum tolerable doses, whereas only 60% was attained in the human cell strain.

Discussion
There is a striking difference in Cd tolerance between cultured cells, either human or murine, previously being adapted to 100 pmole Cd/L culture medium and the parent cells not previously exposed to Cd. The critical level of intracellular Cd in the parent cells described is about 6 nmole/ mg cell protein, whereas the Cd-adapted strains grow continuously at levels 4 to 7 times higher. In the resistant strains, 95% of the intracellular Cd is bound to MT, as determined by gel filtration elution profiles. Thus the level of Cd not bound to MT is well below the level which is lethal to the parent cells. It is suggested that the binding of Cd to MT also explains the slow release of Cd from these cells compared to the parent cells, when Cd is removed from the medium. Bakka et al. (12) also used another method to study the binding modes of Cd in cells, namely, the spectroscopic technique of perturbed angular correlation of yrays (PAC). As far as we know, this is the first time PAC has been used for this purpose (13) in living cultured cells. Those studies confirm the conclusion that there are two different types of Cd binding sites within cells. MT constitutes the main one, binding >60% of the Cd. Additional important information is that this MT apparently is freely suspended in the cytoplasm of the cells. Thus, there is good evidence that MT is one main protecting factor against the toxic Cd ion in these cells and that mechanisms leading to decreased concentration of Cd in the cells does not seem to be at work. This is in agreement with the suggested role of MT regarding resistance against gold (III) chloride. The fact that gold binds to the pre-existent Cd-induced MT in the Cd-resistant human epithelial cell strain probably protects cells from the otherwise lethal gold concentration (4). Also the study on the effect and toxicity of chelating agents led to the assumption that Cd exists in at least two pools within the cells. One rather small part of the Cd is relatively loosely bound and can easily be removed within a few hours by a chelating agent. The other is tightly bound within cells and is removed slowly. It seems plausible to suggest that the small, loosely bound fraction represents a more toxic threat to the cells than does the major pool, which probably comprises the MT-bound Cd. An efficient chelating agent should preferably be able to release Cd from both pools. This capacity will be dependent on the relative affinities of Cd to the chelator versus the MT. None of the compounds tested appeared to have the capacity of releasing more than minor amounts of Cd bound to MT. It appears reasonable to test new chelators in systems including MT, and the in vitro system described by Bakka et al. (10) seems to be a promising system for primary screening of effect and toxicity of such agents. The high toxicity of BAL has also been demonstrated by others in studies on mice (14). The superior releasing effect of MSA in vitro calls for further investigation in vivo.
The demonstrated stability ofthe Cd-resistance in previously Cd-treated cell strains of both human and murine origin might be due to their capacity of immediate de novo MT synthesis when re-exposed to the Cd ion. This has also been demonstrated by in vivo studies, in which the murine cells were grown as tumors in athymic nude mice, that is, in the absence of Cd, and then re-exposed them to Cd after plating the tumor cells either in tissue culture flasks or in soft agar (11). This mechanism of preserving the acquired Cd resistance is compatible with the evidence presented by Hildebrand et al. (15) regarding Cd resistance in cultured CHO cells. In terms of genetics, the explanation for the difference in capacity for de novo MT synthesis between the murine and human cell strain is not yet known. One might speculate that differences in degree of amplification of the MT gene account for this difference. It is reported (16) that gene amplification occurs in Cd-resistant Friend leukemia cells, when compared to their parent nonresistant cells.

Conclusions
Studies on Cd uptake and metabolism in cultured murine and human cell strains with and without Cd-induced MT have revealed the following.
(1) The amount of non-MT-bound Cd seems decisive for Cd toxicity in cells. The critical level seems to be about 6 nmole/mg cell protein in three different cell lines. The resistant cells can tolerate total amounts of intracellular Cd which are several times higher, if the major part is bound to MT, and the pool of non-MT-bound Cd does not exceed this critical level.
(2) The PAC technique is well suited to study binding modes of Cd in living cells and indicates that the cellular Cd is mainly bound to MT and that Cd-containing MT molecules are freely suspended in the cell cytoplasm.
(3) An in vitro model of cultured cells containing MT seems to be a promising test system for primary screening of effect and toxicity of new chelators. Chelating agents apparently remove the non-MT-bound Cd pool in cells. MSA seems more efficient and less toxic than BAL.
(4) The stability of Cd resistance varies between human epithelial cells and mouse fibroblasts in culture. The evidence presented is in agreement with the concept that resistance to the potent MT-inducing metal Cd is stable due to the capacity of cells for de novo synthesis of MT shortly after re-exposure to the metal, both in vitro and in vivo.