Cadmium, an effector in the synthesis of thionein.

Cadmium can elicit the synthesis of thionein in liver cells independent of tissue-organ interactions. The metal diffuses across the plasma membrane and is partitioned between subcellular components in a time dependent manner such that thionein synthesis responds to levels of nonspecifically and specifically bound cytoplasmic metal. Cadmium appears to function at the transcriptional level, and the metal may act to increase the pool of specific m-RNA's.


Introduction
Cadmium is among a group of closely related elements (Cu, Zn, Cd, and Hg) which have been isolated in animals in association with sulfur-rich metal-binding proteins (1)(2)(3)(4)(5)(6)(7). Animals respond to short-term sublethal levels of the metals by producing increased quantities of these specific cytoplasmic proteins known collectively as the metallothioneins (5)(6)(7)(8)(9)(10)(11)(12)(13). Neither the mechanisms which produce and regulate this response nor the precise role metals play in mediating it are clear. Cadmium is among the most toxic elements in the group (14,15) and is also very effective in eliciting the production of metallothionein in liver (11,13). An understanding of the mechanisms which enable organisms to respond to heavy metal exposures, and in particular to cadmium, is essential for evaluating the effects of environmental pollution by heavy metals.
We have previously suggested that the synthesis of thionein may be regulated in liver by cadmium acting as an extracellular effector (16)(17)(18)(19)(20), according to a model which can be summarized as follows. The metal induces thionein in the absence of synergistic and antagonistic effects between different tissues and organs. Cadmium enters liver cells by diffusing across the plasma membrane, and is partitioned between cytoplasmic and nuclear components such that the uptake and disappearance of cadmium from nuclei can be correlated with the ap-* Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70122. pearance of cytoplasmic Cd-thionein. Cadmium appears to act at the transcriptional level in liver, and cadmium may function to increase the pool of translatable thionein m-RNA's.
We believe that there is a growing body of evidence to support these points and a summary of some of the evidence follows. However, we wish to stress that it is not yet known whether cadmium functions directly, or in association with other molecules or ions.

Materials and Methods
Adult male Sprague-Dawley rats weighing 250-300 g were housed in galvanized steel cages, had free access to Purina Rat Chow (Ralston Purina Co., St. Louis, Mo.) and glass-distilled water.
Analytical-grade cadmium chloride and other metal salts were from Baker Chemical Co. (Phillipsburg, N. J.). Male Jax mice of the H/J strain were housed in polypropylene cages and were further treated as described earlier (20). '15Cd (0.132 mCi/mg) and [35S]-cystine (92 mCi/mg) were from New England Nuclear Corp. (Boston, Mass.); actinomycin D, and cycloheximide were from Sigma Chemical Co. (St. Louis, Mo.).
The isolation of cadmium thionein and the measurements of cadmium and 35S-cysteine were carried out as described earlier (19,20). The methods for cell isolation (rat liver and mouse tumor) as well as their treatment with cadmium, preparation of thionein, and its measurement have been fully described (19,20). The isolation and translation of February 1979 281 Volume (ml) FIGURE 1. Cadmium-thionein from rat liver cc pensions were exposed for 15 min to 1.0,g g unlabeled for the cysteine experiment), an bated as described (19)  polysomes from control and cadmiur were carried out essentially as describ and Cousins (21).

Results and Discussion
Cadmium Appears to Function Induction in the Absence of Ti Interactions Synthesis of Cd-thionein occurs rea response to whole body exposure to t lated cells in culture retain this ab dently of other tissue-organ interactic for different cell types in several lab 20,[22][23][24]. Our laboratory has studie liver cells and mouse hepatoma cel cultures. When rat liver cells were inc min in Earle's solution containing 1 Cd-thionein fraction was isolated fr( following heat treatment and gel chi (Fig. IA). This thionein fraction fron was separated into two subfractions ( and Cd-thionein 2) by DEAE cellulose chromatography (Fig. IB). These frac from rat liver single cells were simila thionein obtained from in vivo studie4 We have also observed Cd-thioneii primary cultures of tumor cells. Cells mouse hepatomas, upon exposure to c ,ug/ml), incorporated the metal into a tified as Cd-thionein by gel filtration and ion-_-m O exchange chromatography ( Fig. 2A, B). The ap---4005M pearance of Cd-thionein in tumor cells was accompanied by the incorporation of radioactive cysteine into the protein (Fig. 2C) reparations were between Subcellular Components ) The cadmium-

Lbove experiment
In isolated cells exposed to 1 ,ug Cd/ml, cadmium 7), and then fracuptake did not vary significantly with time of incubation; however, if the cadmium concentrations were varied metal uptake increased linearly with -treated .rats increasing cadmium concentrations (19). These obed by Squibbs servations suggested that the cells absorbed the metal by diffusion, an equilibrium was established fairly rapidly after exposure, and the higher the concentration to which cells were exposed, the in Thionein more metal they absorbed (19).
in Thionein Once the metal is inside the cell, it is thought that issue-Organ cadmium is distributed among subcellular components in a time dependent fashion as described below. Initially, after an acute cadmium exposure idily in vivo in (20 ,tmole/kg), cadmium binds nonspecifically to ;he metal. Iso-high molecular weight proteins; this type of binding ility indepen-decreases with time and at 24 hr is less than 5%. In )ns, as shown contrast, the level of specifically bound metal inoratories (19, creases progressively, and at 24 hr more than 90% is .d isolated rat associated with Cd-thionein. Cadmium enters the lls in primary nucleus following cell exposure to the metal, estabcubated for 15 lishes an equilibrium, and decreases thereafter as Ag Cd/ml, a the amount of thionein-bound metal in the cytocm the lysate plasm increases. Thus, there is a strong correlation romatography between the appearance and binding of the metal to i gel filtration thionein, and the uptake and disappearance of cad-(Cd-thionein 1 Environmental Health Perspectives 282 mium from the nucleus. It is possible for metal contamination to arise from isolation procedures such that the observed cytoplasmic and nuclear metal levels may not correspond to endogenous levels. This possibility was investigated in the following experiment: homogenates from control (no cadmium) and experimental (10,uCi 115Cd/5 gmole of Cd) animals were subjected to a 700g centrifugation, and the resulting cytoplasmic and crude nuclear pellets were removed, divided into several equal aliquots, and reconstituted. Control nuclei were combined with experimental cytoplasm, experimental nuclei with control cytoplasm, and experimental nuclei with experimental cytoplasm. Samples were incubated at 0-40C for 1 hr with stirring prior to isolation of purified nuclei, and an analysis of the nuclei is given in Table 1.
Little or no metal entered control nuclei from the experimental (Cd-thionein containing) cytoplasm, and neither did metal "leak" from experimental nuclei upon exposure to control cytoplasm. It appears that 20 and 24 hr after an acute cadmium exposure the subcellular partitioning of cadmium is a measure of endogenous levels of metal and not an artifact of the system.

Action of Cadmium in Isolated Hepatocytes
In an isolated single cell system the chemical environment, and in particular the amount of metal to which the cells are ultimately exposed, are under the control of the experimenter. Independent studies on the kinetics of induction and the appear- ance of Cd-thionein in whole animals have shown that maximum thionein synthesis, after an acute cadmium exposure, appears to be preceded by a lag period of about 2 hr and to continue for approximately 8 hr. When a similar time study was performed with isolated cells exposed to 1 ,ug of Cd/ml during the preincubation, the cells incorporated cadmium into thionein continuously up to last time measured (6-8 hr). We believe the increase between 0 and 2 hr was due to thionein synthesis and not only to zinc-displacement in thionein (19).
In protein synthesis inhibitor experiments in which variables of concentration and time of exposure were controlled, cycloheximide and actinomycin D were added at either zero time (the end of cadmium exposure), or two hours thereafter. Under these conditions, cycloheximide stopped thionein synthesis at zero and 2 hr (Fig. 3). When actinomycin D was added at time zero, it also prevented any apparent increase in thionein. However, if actinomycin was added 2 hr after thionein synthesis had started, it was only partly effective in inhibiting thionein synthesis. It is probable that enough mRNA's were synthesized between 0-2 hr to maintain a limited synthesis of thionein after inhibition of transcription.
Time of CellI Harvest (Hours) FIGURE 3. Effects of actinomycin-D and cycloheximide on cadmium-thionein synthesis. Rat liver cell suspensions (50 mg/ml) were incubated for 15 min in Earle's salt solution containing I ,zg of radioactively labeled ('15Cd) cadmium per ml. Cells were incubated in Eagle's medium and, at the times indicated, harvested for cadmium-thionein isolation. Actionomycin D and cycloheximide were added at the beginning (t = 0), or 2 hr (t = 2) after incubation had started to a final concentration of 5 ,ug per ml. The bars, solid bracketed lines, and the numbers in parentheses represent the mean ± SEM (n), respectively. mental) animals, and show in a cell-free homologous system that cadmium-thionein synthesis is regulated by changes in the pool of translatable thionein mRNA, as reported by Squibb and Cousins for Zn-thionein (21). Experiments carried out in our laboratory over the past months have not conclusively demonstrated differences between control and experimental polysomal activity in a cell-free translation system. Cherian and Goyer (25) have also noted the failure to develop successfully in vitro systems to study the biosynthesis of metallothionein. Thus, to date, in vitro translation experiments have not confirmed the in vivo effects of cadmium on RNA metabolism (18,24,26,27), and in particular the stimulated synthesis of messenger RNA's following cadmium exposure (24). The detailed mechanisms which regulate cadmium-thionein synthesis are unknown at the present time; however, it is clear that in rat liver (1) they involve controls at the transcriptional level, (2) these controls are responsive to the levels of nonspecifically bound cadmium in the cell, (3) synthesis of thionein decreases as the cytoplasmic levels of this protein sequester intracellular cadmium. An understanding of the regulatory processes of thionein synthesis and degradation is needed to assess how excessive doses of heavy metals can cause permanent damage, or even death, in organisms. This work was supported by National Institute of Health Research Grant ROI-ES00802 from the National Institute of Environmental Health Sciences (USA). The authors thank Mr. Walter Birdsall for the generous gifts of tumor-bearing mice and for excellent technical assistance.