Role of extracellular zinc and copper on metallothionein regulation in cultured rat hepatocytes

Abstract

Cellular and extracellular metallothionein contents of rat hepatocytes cultured in the presence of albumin (30 pmoVL) with zinc (1,10,50 and 100 pmol/L), copper (1, 10 and 50 pmol/L), zinc and copper (1, 10 and 50 pmol/L of each metal) or no metals in the culture medium have been measured by radioimmunoassay. Cellular metallothionein levels increased steadily with culture time regardless of the metal treatment and showed little dependence (only a twofold increase) on extracellular zinc or copper at all metal concentrations and times (up to 3 days) studied. In contrast, the presence of both metals simultaneously in the culture medium strongly increased cellular metallothionein contents, acting synergistically in some cases. Significant extracellular metallothionein was observed when copper or zinc and copper were present in the culture medium, most of which is likely a consequence of cell leakage because no evidence of physiological secretion was observed. Total metallothionein production (cellular and extracellular metallothionein levels) indicated that copper was a better metallothionein inducer than zinc in these experimental conditions. These results indicate that metallothionein regulation in the hepatocyte is different depending on the extracellular metal levels and composition and that attention must be given to metallothionein release from the hepatocyte. (HEPATOLOGY 1991;14:648-654.)The liver is believed to be a central organ in zinc (Zn) and copper (Cu) homeostasis in the organism (1, 2). To characterize how the liver regulates the metabolism of these essential heavy metals, the use of cultured hepatocytes has proved to be a useful tool. Several comprehensive studies on the hepatocyte metabolism of Zn and Cu have been published (3-11). In these studies metallothionein (MT), a low molecular weight heavy metal-binding protein, was usually considered to play a central role. However, its levels were either not measured or measured by rather insensitive or nonspecific The putative secretion of MT by cultured hepatocytes has never been studied in detail. This is noteworthy because experimental studies suggest a normal presence of circulating MT (12-21), the source of which, at least in part, might be the liver (22). Biliary excretion of MT has also been reported (16, 23). Studies on the putative secretion of MT by hepatocytes therefore are needed to establish such a possibility.Therefore the aim of this work was to study the effect of physiological (10 p,moliL) and nonphysiological (0, 1, 50 and 100 FmoVL) concentrations of Zn and/or Cu in the culture medium (in the presence of the physiological chelator albumin) on hepatocyte MT content and the putative MT secretion in those conditions (the terms physiological and nonphysiological for metal concentrations are relative; see Discussion). MATERIALS AND METHODSIsolation and Maintenance of Hepatocytes. Isolated liver parenchymal cells were prepared from adult male Lou rats by using the three-step perfusion technique of Garvey and Heil (24) as modified from Seglen ...

Copper overload affects copper and iron metabolism in Hep-G2 cells

“…Our results indicate that increases in the cellular Cu concentration are not directly proportional to extracellular Cu-His exposition. Although the addition of 10 mol/l Cu-His resulted in an apparently significant increase in cellular Cu, further increases in Cu-His in the media induced MT expression, as expected (26,39,50), but did not result in additional increases in Cu content. Considering that in our experimental condition cells reached a plateau at 5-10 M both in 64 Cu uptake and Cu content, we propose that the capacity of Hep-G2 to accumulate Cu reaches a plateau near the physiological concentration of the metal.…”

“…In the cytoplasm, free Cu rarely exists, because it can readily generate free radicals or oxidize cellular components through its redox activity (4,17). Therefore, Cu is immediately transferred to glutathione (GSH) and metallothionein (MT); the latter is a cytosolic protein that binds up to 12 atoms of Cu and increase its cellular level in response to Cu exposure (5,26,38,41,44). GSH and MT, along with Cu chaperones, provide efficient and specific mechanisms for safe intracellular storage and transport of this metal (25,35).…”

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“…2001). MT is known to act on both the intra‐ and extracellular environment by regulating the levels of heavy metals and by scavenging free radicals (Hidalgo et al. 1991).…”

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“…64 Cu uptake in Cu-deficient Hep-G2 cells showed a twofold decrease in Km compared with cells grown in 10 M Cu. Therefore, Cu is immediately transferred to glutathione (GSH) and metallothionein (MT); the latter is a cytosolic protein that binds up to 12 atoms of Cu and increase its cellular level in response to Cu exposure (5,26,38,41,44). Cells treated with Ͼ50 M Cu, showed an eightfold increase in cytosolic metallothionein.…”