The present paper centers on mammalian metallothionein 1 and 2 in relationship to cell and tissue injury beginning with its reaction with Cd²⁺ and then considering its role in the toxicology and chemotherapy of both metals and non-metal electrophiles and oxidants. Intertwined is a consideration of MTs role in tumor cell Zn²⁺ metabolism. The paper updates and expands on our recent review by Petering et al. (Met Ions Life Sci 5:353-398, 2009).

It has been reported that Zn7-metallothionein (MT), contains one weak binding site for Zn2+. To test this conclusion, rabbit liver MT isolated at pH 7 was reacted with chelating agents of modest affinity for Zn2+. Contrary to the previous study, no evidence was found for Zn2+ stoichiometically bound to the protein with an apparent stability constant of about 108. Indeed, stability constant measurements based upon competition between Zn7-MT and ligands of known stability with Zn2+ showed that all of the protein bound Zn2+ displayed the same stability constant at pH 7.4 and 25° C of (1.7±0.6) × 1011. Brief reaction of Zn7-MT with strong acid converted it into MT* and upon reneutralization into Zn7-MT*, which demonstrated reactivity of about 1 Zn2+/mol MT with competing ligands. Acid titration of Zn7-MT to pH 2 or below rapidly resulted in the formation of Zn7-MT* that displayed biphasic titration with base, revealing the rebinding of lower affinity Zn2+ between pH 5 and 7. Since MT is commonly acidified during preparation, care must be taken to document which form of the protein is present in subsequent experiments at pH 7.

The hypothesis was tested that Cd2+ undergoes measureable reaction with the Zn-proteome through metal ion exchange chemistry. The Zn-proteome of pig kidney LLCPK1 cells is relatively inert to reaction with competing ligands, including Zinquin acid, EDTA, and apo-metallothionein. Upon reaction of Cd2+ with the Zn-proteome, Cd2+ associates with the proteome and near stoichiometric amounts of Zn2+ become reactive with these chelating agents. The results strongly support the hypothesis that Cd2+ displaces Zn2+ from native proteomic binding sites resulting in the formation of a Cd-proteome. Mobilized Zn2+ becomes adventitiously bound to proteome and available for reaction with added metal binding ligands. Cd-proteome and Zn-metallothionein readily exchange metal ions, raising the possibility that this reaction restores functionality to Cd-proteins. In a parallel experiment, cells were exposed to Cd2+ and pyrithione briefly to generate substantial proteome-bound Cd2+. Upon transition to a Cd2+ free medium, the cells generated new metallothionein protein over time that bound most of the proteomic Cd2+ as well as additional Zn2+.