Frank O. Brady

The physiological function of metallothionein

Abstract Metallothionein seems to provide zinc and copper to newly synthesized apoenzymes in tissues which are undergoing rapid growth and development. High concentrations of this protein are also induced in the liver by external physical and internal chemical stress and when the liver regenerates after partial hepatectomy. Metallothionein, thus, has a central role in macromolecular synthesis.

Synthesis of rat hepatic zinc thionein in response to the stress of sham operation

Abstract Following sham operation for adrenalectomy, a dramatic 30-fold increase in rat hepatic zinc thionein occurs, peaking at 18 hours after surgery. Hepatic cytosolic and serum zinc levels rise concomitantly with zinc thionein. Copper in hepatic thionein and cytosol rises only slightly and serum copper not at all during the period of observation. In the period 18 to 48 hours after surgery the content of hepatic zinc thionein decreases with a t 1 2 of 16.4 hours. Pretreatment with cycloheximide (1 mg/kg b.w.) two hours before surgery inhibits the rise in zinc thionein by 52%, the rise in cytosolic zinc by 56%, and actually causes a decrease in serum zinc by 33%. Pretreatment with the α-adrenergic receptor blocker, phetolamine (10 mg/kg b.w.), or the β-adrenergic receptor blocker, propranolol (10 mg/kg b.w.), 30 minutes before surgery also inhibited the rise in zinc thionein (82% and 60%, respectively) and cytosolic zinc (75% and 47%, respectively), and decreased serum zinc (38% and 44%, respectively) 19 hours after surgery. Treatment with corticosterone (40 mg/kg b.w.) alone or epinephrine (1–20 μg/kg b.w.) alone did not alter hepatic zinc thionein levels 18 hours late, although they each caused hypozincemia and epinephrine raised cytosolic zinc levels. Treatment with corticosterone and epinephrine together did, however, raise zinc thionein levels 3.2-fold (P These experiments are consistent with the hypothesis that adrenal hormones are involved in the regulation of zinc metabolism, and, hence, zinc thionein in levels in rat liver following the stress of sham operation.

Displacement of zinc and copper from copper-induced metallothionein by cadmium and by mercury: in vivo and ex vivo studies.

The in vitro affinity of metals for metallothionein (MT) is Zn less than Cd less than Cu less than Hg. In a previous study Cd(II) and Hg(II) displaced Zn(II) from rat hepatic Zn7-MT in vivo and ex vivo (Day et al., 1984, Chem. Biol. Interact. 50, 159-174). The ability of Cd(II) or Hg(II) to displace Zn(II) and/or Cu(II) from metallothionein in copper-preinduced rat liver (Zn, Cu-MT) was assessed. Cd(II) and Hg(II) can displace zinc from (Zn, Cu)-MT both in vivo and ex vivo. The in vitro displacement of copper from MT by Hg(II) was not confirmed in vivo and ex vivo. Cd(II) treatment did not alter copper levels in (Zn, Cu)-MT, as expected. Hg(II) treatment, however, did not decrease copper levels in MT, but rather increased them. The sum of the copper increase and mercury incorporation into MT matched the zinc decrease under in vivo conditions and actually exceeded the zinc decrease under ex vivo conditions. Short-term exposure of rat liver to exogenous metals can result in incorporation of these metals into MT by displacement of zinc from pre-existing MT. Displacement of copper from pre-existing MT by mercury, as predicted by in vitro experiments, was not confirmed under the conditions of our in vivo and ex vivo experiments. This result is explainable based on the differing affinities and/or preferences of the two metal clusters in MT.

In vivo and ex vivo displacement of zinc from metallothionein by cadmium and by mercury.

Divalent cadmium and mercury ions are capable in vitro of displacement of zinc from metallothionein. This process has now been studied in vivo and ex vivo, using the isolated perfused rat liver system, in order to determine if this process can occur in the intact cell. Rats with normal and elevated (via preinduction with zinc) levels of hepatic zinc thionein were studied. Cd(II) completely displaces zinc from normal levels of metallothionein and on a one-to-one basis from elevated levels of metallothionein, both in vivo and ex vivo. Hg(II) displaces zinc from metallothionein (normal or elevated) rather poorly, as compared with Cd(II), in vivo, probably due to the kidneys preference for absorbing this metal. Ex vivo Hg(II) displaces zinc from metallothionein (normal or elevated) on a one-to-one basis, with considerably more mercury being incorporated into the protein than in vivo. The results of double-label ex vivo experiments using metal and [35S]cysteine (+/- cycloheximide) were consistent with the above experiments, indicating that de novo thionein synthesis was not required for short term incorporation of cadmium and mercury into metallothionein. These data are supportive of the hypothesis that cadmium and mercury incorporation into rat hepatic metallothionein during the first few hours after exposure to these metals can occur primarily by displacement of zinc from preexisting zinc thionein by a process which does not require new protein synthesis.

In vivo and ex vivo effects of copper on rat liver metallothionein

Abstract In order to test the hypothesis that exogenous copper could displace endogenous zinc from metallothionein under in vivo and ex vivo conditions, a series of zinc-copper competition experiments were conducted with rat liver. Intraperitoneal injection of zinc alone resulted in a large increase in zinc and a small increase in copper associated with hepatic metallothionein 24 hr later, as compared to controls. Intraperitoneal injection with copper alone resulted in increases in both zinc and copper bound to metallothionein 6 hr later. Intraperitoneal injection of zinc at time zero and copper at 18 hr, followed by killing at 24 hr, resulted in no increase in zinc, but a significant increase in copper, bound to metallothionein. Exposure of rat livers to copper ex vivo in an isolated perfusion apparatus gave different results from the in vivo experiments in regards to the metal composition of metallothionein. Animals were injected ip with zinc at time zero, and their livers were removed at 18 hr for a 2-hr perfusion. The zinc and copper contents of metallothionein in the groups receiving zinc in vivo and then either killed or perfused without additional metal were the same. The group which received zinc in vivo and copper ex vivo showed a large decrease in its metallothionein zinc content, a decrease which was matched almost exactly by a large increase in its metallothionein copper content. These results are consistent with the above-mentioned hypothesis and with recent in vitro results demonstrating the ability of copper to displace zinc from metallothionein [D. Holt, L. Magos, and M. Webb, Chem.-Biol. Interact. 32, 125 (1980)].

Radioimmunoassay of rat liver metallothionein.

Abstract A simple, inexpensive and convenient radioimmunoassay for rat liver metallothionein has been developed. The double-antibody assay involves the labeling of homogeneous, rat liver zinc thionein with trace amounts of 109 Cd(II) to a specific activity of 1–2 × 10 6 cpm/μg protein; the binding of this antigen by rabbit anti-rat liver metallothionein antiserum; the displacement of this antigen by unlabeled zinc thionein or cadmium, zinc-thionein; the precipitation of the rabbit antibody-rat antigen complex by goat anti-rabbit IgG immunoglobulins; and the binding of this precipitate to cellulose acetate filters. The radioimmunoassay is useful in the range of concentration of metallothionein of 10–500 ng protein. The assay is accurate as compared with a previous technique of quantitating metallothionein in extracts of rat liver. A radial immunodiffusion assay for metallothionein is also described.

Induction of zinc metallothionein by calcium ionophore in vivo and in vitro

The calcium ionophore, A23187, can induce rat hepatic metallothionein (MT) when administered in vivo (5.8‐fold, 5.0 μM, 11 h) and rat hepatocyte MT when administered in vitro (10.70‐fold, 1.0 μM, 24 h). Several rat hepatoma cell lines (2M, 4.55‐fold; JM2, 12.29‐fold; EC3, 14.12‐fold; HTC, 7.99‐fold) and a normal rat liver cell line (Clone 9, 39.67‐fold) were tested for their inducibility of MT mRNA by Cd2+ (10 μM, 8 h). Quantitatively, JM2 and 2M made the most MT mRNA, while HTC made the least. A23187 (0.1–7.0 μM) was studied as an inducer of MT mRNA in these cell lines (except for HTC) and in HeLa. A variety of responses and tolerances were seen with inductions ranging up to 32.11‐fold. Quantitatively, the best responding cell lines were EC3 and 2M. A combination induction experiment, using TPA, a protein kinase C activator, and A23187 in EC3 cells revealed an additive effect of the two inducers on MT mRNA levels: TPA (10 nM), 11.71‐fold; A23187 (3.0μM), 6.71‐fold; and TPA + A23187, 20.00‐fold. These studies have implicated perturbations in cytosolic calcium ion concentrations, caused by the ionophore A23187, as being involved in the complicated signaling systems which can lead to induction of MT mRNA and protein.

Inhibitors of Ca2+ channels, calmodulin and protein kinases prevent A23187 and other inductions of metallothionein mRNA in EC3 rat hepatoma cells

The role of calcium in the induction of MT mRNA has been studied in EC3 rat hepatoma cells, using various inducers (A23187, TPA, norepinephrine, and 2-chloroadenosine) and inhibitors (H7:PK-A and PK-C; W7:calmodulin; verapamil:calcium channel blocker; and TMB-8; cytosolic calcium chelator). The inhibitions of inductions observed in this study were consistent with calcium playing an important role in MT mRNA induction by itself and via crosstalk among the PK-A, PK-C, and calmodulin-dependent protein kinase pathways. Calcium has an important role in the complicated second messenger pathways which result in the positive interaction of transcription factors with the promoters of MT genes.

The effect of adrenalectomy on zinc thionein levels in rat liver.

Abstract The concentrations of zinc thionein and cytosolic zinc in rat liver were examined in male rats five days after bilateral adrenalectomy. Zinc in metallothionein increased 10 fold, as compared with control animals. Cytosolic zinc increased 79% as compared with controls. 65% of this increase could be accounted for bound to metallothionein. Sham operated animals after five days showed a 4 fold increase in hepatic zinc thionein and a 23% increase in cytosolic zinc, 71% of this increase being bound to metallothionein. Adrenalectomized rats, maintained on daily injections of corticosterone (4mg/100g b.w.), exhibited the same levels of zinc thionein and cytosolic zinc as adrenalectomized rats receiving no treatment. Adrenalectomized rats, maintained on daily injections of aldosterone (5μg/100g b.w.), exhibited the same levels of zinc thionein as the sham operated rats, but the cytosolic zinc remained elevated at the level found in adrenalectomized rats receiving no treatment. These results indicate that there is adrenal involvement in the control of hepatic zinc and zinc thionein levels in the rat.

Postinductive actinomycin D effects on the concentrations of cadmium thionein, and copper chelatin in rat liver☆

The time courses of induction in rat liver of copper chelatin by copper, cadmium thionein by cadmium, and zinc thionein by copper, cadmium, and zinc were monitorg metal were used in order to avoid toxic effects, being 5 mg zinc, 0.5 mg copper, and 0.25 mg cadmium per kg body weight. Peak times of induction and half times of decay observed were: copper chelatin (9 h, 8.6 h), cadmium thionein (18 h, 6.80 days), and zinc thionein (zinc rats, 18 h, 10.1 h; copper rats, 9 h, 18.2 h; cadmium rats, 24 h, 4.53 days). Administration of actinomycin D (1 mg per kg body weight) at the peak times of induction of the various proteins had no effect on the concentrations of chelatin or cadmium thionein observed up to 24 hours later, but in the case of zinc thionein, induced by zinc, copper, or cadmium, elevated concentrations were observed up to 23 h after administration of the drug. Such behavior is reminiscent of superinduction previously seen with other proteins and enzymes. We postulate that the intracellular concentration of free zinc in liver is of fundamental importance in the induction of zinc thionein, and this can be distributed by exogenous copper or cadmium resulting in the induction of synthesis of zinc thionein.