Expression of Metallothionein in the Liver of Rats Fed Copper-Deficient AIN-93G Diet

Copper-loading induces metallothionein (MT) synthesis in the liver of rats. Copper-MT from the liver of copper-injected rats has been isolated and analyzed. The copper-induced MT synthesis is characterized by increased incorporation of [ 35S]cysteine into the protein and enhanced production of the MT mRNA, suggesting that copper induction of MT occurs at the level of gene transcription. The mechanism by which copper induces MT synthesis, however, remains unsolved. It is also unknown whether copper is essential for MT production under a diversity of physiological and pathological conditions. The purpose of this study was thus to examine the effect of dietary copper deficiency on MT induction in rats. A copper-deficient diet was formulated by replacing copper with the corresponding weight of corn starch in the All'J-93G diet. Diet analysis for copper yielded values of 5.7 mg Cu/kg diet for the copperadequate diet and 0.4 mg Cu/kg diet for the copper-deficient diet. Male, weanling Sprague-Dawley rats (46-57 g; Sasco, Lincoln, NE, USA) were divided into two weight-matched groups having average weights of 52 g each; one had free access to the copper-adequate diet and the other to the copper-deficient diet. They also had free access to deionized water. After 4•weeks on their respective diets and an overnight fast, each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (65 mg/kg body wt, Vet Labs, Lenexa, KS). Blood was withdrawn from the inferior vena cava for erythrocyte counting and plasma assays. The liver and heart were removed, flushed with cold 0.9% NaCl via their major vessels and divided for subsequent assays. Tissue samples including the liver, heart, and kidney were stored at —20°C for mineral assays and those for MT and mRNA assays were placed in liquid nitrogen, then stored at —80°C. Hematocrit and hemoglobin content were determined on a cell counter. An automated analyzer was used to determine serum ceruloplasmin. Trace element contents of tissues were determined by inductively coupled argon plasma emission spectroscopy after lyophilization and digestion of the tissues with nitric acid and hydrogen peroxide. A routine Northern blot assay was used to analyze MT-I mRNA. A probe corresponding to a 1185-base pair Hind HI and Bgl 11 fragment of mouse MT-I cDNA was used to identify the MT transcript on the membrane. Autoradiographic images were scanned and analyzed by an imaging analyzing system. Densitometric values were then determined from digitized images of autoradiograms. MT concentrations were measured by the Cd/hemoglobin radiometric assay. Characteristics of rats fed the copper-deficient diet were compared to those of rats fed the copperadequate diet. Copper concentrations were significantly (p <0.01) depressed in the liver, heart, and kidney of copper-deficient rats. Zinc concentrations were significantly (p <0.01) decreased in the heart and kidney, but not significantly (p > 0.05) reduced in the liver, lion concentrations were also significantly (p <0.01) depressed in the heart and kidney, but significantly (p <0.01) elevated in the liver. Other changes including reduced activity of ceruloplasmin in the plasma, depressed Cu,Zn-SOD in the tissues, and decreased hematocrit and hemoglobin concentrations in the blood were found in the rats fed the copper-deficient diet, typically indicative of severe copper deficiency.