Two controversial facets of metallothionein biochemistry and toxicology center on the existence of measurable concentrations of apometallothionein in cells and the hypothesis that Cdmetallothionein is the species which causes toxicity to the kidney proximal tubule [1, 2]1 This contribution addresses both issues.

The effects of Cd2+and Cd–metallothionein on two cultured cells with proximal tubule characteristics, mouse kidney cortical cells and pig kidney LLC-PK1cells, have been compared. Cd2+inhibits Na+–glucose cotransport in LLC-PK1cells and in the process decreases the number of binding sites for [3H]phlorizin, a competitive inhibitor of glucose for the Na+–glucose cotransporter. During 24 hr incubation and over a range of concentrations in the two cell types, only Cd2+inhibited Na+–glucose cotransport even when approximately equal concentrations of intracellular Cd resulted from these treatments. Indeed, at low concentrations of Cd–metallothionein in mouse cells, transporter activity was elevated. Extension of incubations to 72 hr in mouse cells led to increased Cd uptake and reduction in cell density with both sources of Cd but only a progressive decline in Na+–glucose cotransport activity with Cd2+. Zn–metallothionein was without effect under comparable conditions. Both forms of Cd were accumulated by these cells, with the large majority of the metal ion localizing in metallothionein as a Cd, Zn–protein in LLC-PK1cells. Under equal exposure conditions, the net uptake of Cd from Cd2+was an order of magnitude greater than Cd from Cd–metallothionein in the two cell types. It is evident that the mechanisms of toxicity of Cd2+and Cd–metallothionein as well as their modes of uptake differ in these two cell types.

Properties of the inhibition of Na(+)-glucose cotransport by Cd2+ in mouse kidney cortical cells have been determined. In no case was any inhibition observed before 3 hr. The extent of inhibition was dependent upon both the concentration of Cd2+ and the length of exposure. Kinetic studies showed that metallothionein mRNA induction by Cd2+ was initiated within 1 hr after incubation with Cd2+ began and peaked by 3-6 hr. Metallothionein protein increased more slowly, beginning at 3 hr and continuing for at least 9 hr. The protein had both Cd2+ and Zn2+ bound to it throughout this period. Nevertheless, a pool of nonmetallothionein Cd2+ appeared after 3 hr, coinciding with the onset of inhibition of Na(+)-glucose cotransport, and increased over the next 9 hr. Pretreatment of cells with Zn2+ protected them from the effects of Cd2+ on Na(+)-glucose cotransport. It delayed the onset of inhibition of transport as well as the extent of inhibition. Detailed analysis of the distribution of Cd2+ and Zn2+ in the soluble fraction of these cells showed that the concentration of non-metallothionein bound Cd2+ was not suppressed by the presence of Zn-metallothionein after the onset of exposure to Cd2+. Incubation of cells with larger concentration of Zn2+ and Cu2+ also inhibited Na(+)-glucose cotransport.