0 Pliurmacology & Toxicology 1999, 84, 255-260. Printed in Denmork . AN rights reserved Copyright 0 ISSN 0901-9928 The Effect of Heavy Metal-Induced Metallothioneinon Zn, Cu and Cd Accumulation in Rat Kidney Shigeru Saito', Masashi Okabe2, Katsumi Yoshida' and Masaaki Kurasaki2 'Department of Preventive Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki 21 6-85 11, 'Department of Environmental Medicine and Informatics, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0908, Japan (Received October 5, 1998; Accepted December 17, 1998) Abstract: To examine the role of metallothionein on heavy metal accumulation in kidneys of rats after Zn, Cd or Cu injection, the relative Zn, Cd or Cu-binding capacity of heavy metal-induced metallothionein in renal cytosol of rats after Zn, Cd or Cu injection was determined. The Zn or Cu increment in renal cytosol after Zn or Cu injection was attributable to low and high molecular weight proteins, while most of the Cd increment was attributable to a low molecular weight protein. The low molecular weight, metal-binding protein was identified as metallothionein using a competitive ELISA. There was a close relationship between heavy metal contents in the renal cytosol and metallothionein of all heavy metal- injected rats. In dose-response and time-course studies, approximately 45, 40 and 85% of the Zn, Cu and Cd increments in renal cytosol were bound to metallothionein after Zn, Cu and Cd injection, respectively. Therefore the order of relative binding capacities of Zn, Cu and Cd-induced metallothionein in kidney was determined to be Cd>Zn -Cu. These results suggest that the role of metallothionein in Zn or Cu accumulation in the kidney after Zn or Cu injection is different from that of metallothionein in Cd accumulation in the kidney after Cd injection. Metallothionein is characterized by low molecular weight (6500-7000 Da), high affinity for heavy metals such as Zn, Cd and Cu, high cysteine content and lack of aromatic amino acids (Kagi 1991 & 1993). Mammalian metallo- thioneins usually have a chain length of 61 amino acids in which the sequence positions of the 20 cysteines are strongly conserved (Kagi 1993). All the cysteines are involved in metal binding, and spectroscopic investigations have pro- vided strong evidence for the metals being bound in two distinct clusters (Otvos & Armitage 1980; Vasak & Kagi 1981). A remarkable feature of all metallothioneins is the inducibility by several heavy metals, hormones, cytotoxic agents, various physiological conditions associated with physical or chemical stresses and X-ray irradiation (Kagi 1993). They are believed to be involved in the homeostasis of the cellular concentrations of essential heavy metals such as Zn and Cu, and in the detoxification of heavy metals such as Cd (Kojima & Kagi 1978; Hamer 1986; Kagi & Schaffer 1988). Although the levels of metallothionein and the inducibility after administration of the heavy metals to rats have been demonstrated in adult rat kidney and liver (Eaton et al. 1980; Onosaka & Cherian 1981 & 1982; Waalkes & Klaassen 1985), the renal or hepatic heavy metal metabolism has not been investigated in detail. In order to understand the roles of metallothionein on heavy metal ac- cumulation in renal cytosol of rats, the relative Zn, Cd and Cu-binding capacities of heavy metal-induced metallothion- Author for correspondence: Shigeru Saito, Department of Preven- tive Medicine, St. Marianna University School of Medicine, 2-16- 1 Sugao, Miyamae-ku, Kawasaki 2168511, Japan (fax +81 44 971 8356). ein (the ratio of heavy metal content in metallothionein to heavy metal increment in renal cytosol) after Zn, Cd and Cu injection were determined by dose-response and time- course studies. Materials and Methods Chemicals. Sephadex G-75 was obtained from Pharmacia LKB (Uppsala, Sweden). 0-Phenylenediamine, phosphate-citrate buffer with sodium perborate capsules and avidin-peroxidase were pur- chased from Sigma Chemical Co., USA. Mouse anti-MT IgG (E9: 1st antibody) was from Zymed Laboratories Inc., USA. Sheep anti- mouse IgG biotinylated (2nd antibody) was from Amersham Co., UK. All other chemicals were of commercially available analytical grade. The glassware for acid oxidation was acid-washed and rinsed with deionized and distilled water. Animals. Fifty-four male Sprague-Dawley rats (Japan SLC, Inc., Japan), weighing 125-150 g were divided into 18 groups of 3 rats, housed in constant temperature of 21.5%1.5"and 12-hr lightil2-hr dark cycle for two weeks prior to the experiments. They were pro- vided with a commercial diet (Clea Japan, Inc., Japan) and water ad libitum. The rats were fed a diet of 10 glday, containing 0.64 mg Zn and 0.13 mg CuilO g. In a dose-response study, ZnS04, CuS04 and CdCI2 were dissolved in saline solution to obtain 1 mg (15 pmol), 5 mg (76 pmol), 10 mg (152 pmol) and 20 mg (305 pmol) Znikg, 2 mg (31 pmol), 4 mg (62 p o l ) and 6 mg (94 pmol) Cuikg or 1 mg (8 pmol), 2 mg (17 pmol) and 3 mg (26 pmol) Cdikg, respectively. Zn (20 mglkg), Cu (6 mg/kg) and Cd (3 mg/kg) were maximum tolerated doses. The control rats re- ceived only saline solution. Each rat was injected with a single intraperitoneal injection (0.5 m11250 g rat) of saline or heavy metal doses and killed 14 hr after injection with diethyl ether. Richards & Cousins (1975) have shown that the maximum of in- duced metallothionein content was between 12 and 16 hr after Zn injection. We selected the 14 hr after heavy metal injection. In a time-course study, ZnS04, CuS04 and CdC12 were dissolved in sa-