Induction and Synthesis of Metallothionein in Isolated Perfused Rat Liver*

The induction and synthesis of metallothionein was studied in the isolated perfused rat liver, using Cd(I1) as the inducing metal. Perfusing the liver with 25 to 100 pg of Cd(I1) for 180 min revealed that the amount of Cd(I1) in metallothionein increased with the dosage of Cd(II), saturation being achieved at 75 pg. Zn(I1) in metallothionein decreased to about 60% of the level found in control perfused livers and was not altered thereafter. A time course study [75 pg of Cd(II)] indicated that Cd(I1) incorporation into metallothionein increased with time for the entire 180 min of perfusion. After an initial decrease at 30 min, Zn(I1) in metallothionein increased only slightly. To ascertain whether de novo synthesis of cadmium thionein was being observed, the effects of transcriptional and translational inhibitors on metallothionein synthesis was studied both in vivo and ex vivo. For in vivo experiments, donor rats were pretreated 4 h prior to removal of the liver with intraperitoneal injections of actinomycin D, cordycepin, or cycloheximide, and the livers were subsequently perfused *Cd(II). For en vivo experiments, the inhibitors were added to the perfusion medium 60 min prior to the addition of Cd(I1). Actinomycin D significantly decreased the incorporation of Cd(I1) into metallothionein in vivo; cordycepin and cycloheximide were inhibitory in vivo and ex vivo. Zn(I1) incorporation into metallothionein decreased in all cases. Up to 90% of the Cd(I1) dose was retained by the liver throughout the duration of perfusion, and the rest was secreted into the bile. The latter was dose-dependent and increased when livers were pretreated with the inhibitors. Perfusing with Cd(I1) did not significantly alter the Zn(I1) content of the livers even in the animals receiving inhibitors in vivo. These data indicate that perfusion of the liver with Cd(I1) results in the synthesis of cadmium thionein, and this de novo synthesis is regulated at transcriptional and translational levels.