Anne M. Wood

Effect of zinc, copper and glucocorticoids on metallothionein levels of cultured neurons and astrocytes from rat brain

The knowledge of brain metallothionein (MT) regulation and especially of MT presence in specific cell types is scarce. Therefore, the effect of several well-known MT inducers, measured by radioimmunoassays using antibodies that cross-react with MT-I and MT-II or specific for MT-I and which do not cross-react with human growth inhibitory factor (GIF or MT-III), has been studied in primary cultures of neurons or astrocytes obtained from rat cerebrum. MT-I levels in glial cells were about ten times higher than those in neuronal cells (538 +/- 194 vs. 49 +/- 16 pg MT-I/micrograms protein, mean +/- S.D. from three separate cell preparations). Increasing the concentration of Zn in the bovine serum albumin (BSA)-containing culture medium up to 50 microM significantly increased MT-I levels by up to 3.5-fold in neurons and 2.5-fold in astrocytes. In contrast, Cu up to 50 microM increased MT-I levels in a saturable manner in both neurons (up to 5-fold) and astrocytes (up to 1.5-fold), the maximum effect occurring at 5 microM Cu. In general, the combination of Zn and Cu further increased MT-I levels. The effect of the metals on MT-I appeared to reflect metal uptake, since MT-I induction was less marked when the BSA concentration in the medium was increased from 2 to 10 mg/ml. Dexamethasone increased MT-I levels in both neurons and astrocytes in vitro in a concentration-dependent manner. Endotoxin, IL-1 and IL-6 did not have a significant effect on glial MT levels at the concentrations studied. The administration of dexamethasone to rats increased MT-I levels in non-frontal cortex, cerebellum, pons+medulla, midbrain and hippocampus, but not in hypothalamus, frontal cortex and striatum. Endotoxin increased liver but not brain MT-I levels. Immunocytochemical studies in adult rat brain preparations with a polyclonal antibody that cross-reacts with MT-I and MT-II indicated that immunostaining was always nuclear in glial cells, whereas in neurons it was nuclear in the cerebral cortex, hippocampus and the granular layer of the cerebellum, and nuclear plus cytoplasmic in Purkinje cells in the cerebellum, hypothalamic nuclei and gigantocellular reticular nucleus in the brain stem. Meninges, choroidal plexus, ependymal and endothelial cells were also MT-immunoreactive.

Rat metallothionein-2 containsNα-acetylated and unacetylated isoforms

Mammalian metallothioneins (MT), are characteristically Nα‐acetylated and the presence of an unblocked N‐terminus has not previously been reported. On‐line capillary electrophoresis‐electrospray mass spectrometry of hepatic MT‐2 from rats injected with zinc revealed two isoforms differing by a mass equivalent to that of a single acetyl group. The lower mass component constituted > 20% of total MT‐2 protein and both MT‐2 isoforms were separated by reversed‐phase high‐performance liquid chromatography. The identity of each fraction was confirmed by matrix‐assisted laser desorption ionisation mass spectrometry, and amino acid analysis and N‐terminal sequencing revealed that the lower mass isoform was unblocked at the N‐terminus and had an amino acid composition and sequence which is characteristic of rat MT‐2. Thus the complementary techniques of mass spectrometry and N‐terminal sequencing demonstrated conclusively that purified MT‐2 from zinc‐treated rats contains an unacetylated isoform. We propose that the cotranslational acetylation of rat MT‐2 may under some circumstances be inefficient compared to that in other nonrodent species, where we have detected only trace levels of unacetylated MT isoforms.

The protective role of metallothionein in copper-overload: II. Transport and excretion of immunoreactive MT-1 in blood, bile and urine of copper-loaded rats

The regulation of copper homeostasis in copper overloaded animals occurs by excretion of excess of the metal in bile and urine, which may be facilitated by metallothionein (MT) a copper binding protein. The role of MT in the mobilisation and excretion of copper excess has been studied in copper-loaded rats during the development of tolerance. Young male Wistar rats were fed a high copper (1 g/kg) diet for 16 weeks during which period they were killed after prior collection of bile, blood and urine for analysis for copper and immunoreactive MT-1. In addition bile was separated chromatographically and the eluant fractions were assessed likewise for copper and MT-1. Biliary excretion of copper and MT-1 rose to a maximum after 6 weeks, falling subsequently as the rats became copper tolerant. Early increases in circulating copper and MT-1 occurred likewise but whereas MT-1 fell subsequently during the recovery period, serum copper remained elevated. By contrast, urinary copper and MT-1 maintained an increased output throughout. Chromatographic separation of bile revealed the presence of a range of immunoreactive MT-1 degradation products. It was concluded that the close correspondence between bile and serum MT reflected their hepatic derivation and implicated liver MT as an export protein in the early stages of copper overload. By contrast, urine MT, maintained independently of circulating MT levels, established the active secretory participation of the kidney in promoting the continued depletion of excess copper.

Obesity and hyperleptinemia in a colony of metallothionein (-I and -II) null mice

Mice with targeted disruption of metallothionein (MT)-I and -II genes are reported to have no phenotypic abnormalities and show normal reproduction and development [1, 2]. With the exception of the pancreas, adult tissue Zn levels are largely unaffected by lack of MT-I and -II [3] and retention of Cd by the liver and kidney of MT-null mice after injection of a Cd salt is reduced [4]. Nevertheless, MT-null mice are more sensitive to Cd [5], Cu [6] and Zn [7] toxicity and to oxidant stress [8]. These compounds are also more toxic to MT-null mouse embryo fibroblasts in primary culture [9] and such results are consistent with the proposed roles of MT-I and MT-II in metal detoxification and in scavenging free radicals.

Metallothionein Concentrations in the Blood and Urine of Streptozotocin Treated Rats

The accumulation of metallothionein (MT) in tissues is influenced by changes in Zn and Cu status and also by the occurrence of various types of stress and infection. The effects of these ‘stress’ factors are mediated by changes in hormonal status; glucocorticoids, glucagon and catecholamines can, for example, induce hepatic MT synthesis. In studies of the effects of adrenal and pancreatic hormones on trace metal metabolism, Failla et al. (1985) have shown that hepatic and renal concentrations of Zn, Cu and of MT are greatly increased in rats made diabetic by treatment with streptozotocin (STZ). We have recently suggested that assay of MT in blood and urine can be used in the diagnosis of Zn deficiency (Bremner and Morrison, 1987) but before this technique can be applied in practice more information is needed on the effects of other patho-physiological states on the occurrence of MT in these fluids. It is particularly important that studies be extended to conditions where tissue MT levels are altered. It was therefore of interest to determine the effects of STZ-induced diabetes on MT levels in blood plasma and cells and in urine of rats.

Factors Influencing the Accumulation of Metallothionein in Rat Blood Cells

It has been shown by radioimmunoassay that small amounts of metallothionein (MT) are present in blood plasma and cells of normal rats. Concentrations in plasma generally increase after induction of tissue MT synthesis but little is known of the effects of nutritional and physiological factors on the occurrence of MT-I in blood cells or on its distribution among sub-populations of cells. As part of an investigation into the use of MT assays for the assessment of trace element status, we have examined the effects of zinc deficiency, of zinc and cadmium injection, of endotoxaemia and of induced reticulocytosis on the occurrence of MT-I in blood cells of rats.