No abstract

Many cell types contain metal-ion unsaturated metallothionein (MT). Considering the Zn 2+ binding affinity of metallothionein, the existence of this species in the intracellular environment constitutes a substantial "thermodynamic sink." Indeed, the mM concentration of glutathione may be thought of in the same way. In order to understand how apo-MT and the rest of the Zn-proteome manage to co-exist, experiments examined the in vitro reactivity of Zn-proteome with apo-MT, glutathione (GSH), and a series of common Zn 2+ chelating agents including N,N,N',N'-(2-pyridylethyl) ethylenediammine (TPEN), EDTA, and [(2,2'-oxyproplylene-dinitrilo]tetraacetic acid (EGTA). Less than 10% of Zn-proteome from U87 mg cells reacted with apo-MT or GSH. In contrast, each of the synthetic chelators was 2−3 times more reactive. TPEN, a cell permeant reagent, also reacted rapidly with both Zn-proteome and Zn-MT in LLC-PK 1 cells. Taking a specific zinc finger protein for further study, apo-MT, GSH, and TPEN inhibited the binding of Zn 3 -Sp1 with its cognate DNA site (GC-1) in the sodium-glucose co-transporter promoter of mouse kidney. In contrast, preformation of Zn 3 -Sp1-(GC-1) prevented reaction with apo-MT and GSH; TPEN remained active but at a higher concentration. Whereas, Zn 3 -Sp1 is active in cells containing apo-MT and GSH, exposure of LLC-PK 1 cells to TPEN for 24 h largely inactivated its DNA binding activity. The results help to rationalize the steady state presence of cellular apo-MT in the midst of the many, diverse members of the Znproteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn 2+ .

Observations of apo-metallothionein (apo-MT) have been made under a variety of physiologic circumstances, including zinc deficiency in cell culture and in rodents, cellular induction of MT by dexamethasone with concurrent Zn deficiency, a variety of tumors under normal Zn conditions, MT induction by Zn and Bi citrate, induction of hepatic MT after tumor cell injection into nude mice, and overexpression of cardiac MT in MT transgenic mice. Experiments demonstrating both the lability of Zn and Cu bound to MT and the cellular stability of apo-MT are described to help rationalize the widespread presence of this metal-depleted species. Finally, comparative in vitro and cellular experiments examined the relative reactivity of Zn- and apo-MT with nitric oxide species, showing that apo-MT is much more reactive chemically and that in cells it may be a principal reactive species within the MT pool.

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).

The reactivity of Zn(7)- and Cd(7)-metallothionein (MT) with S-nitrosopenicillamine (SNAP), S-nitrosoglutathione (GSNO), and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) was investigated to explore the hypothesis that metallothionein is a signficant site of cellular reaction of nitric oxide or NO compounds. Zn(7)-MT reacted with SNAP or GSNO only under aerobic conditions and in the presence of light, which stimulates the decomposition of S-nitrosothiolates to NO. Zn(2+) is released, and protein thiols are modified. DEA/NO, which degrades spontaneously to release NO, also reacted with Zn(7)-MT only when oxygen was present. Anaerobically, DEA/NO reacted with Zn(7)-MT in the presence of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, which converts NO to NO(2). Glutathione competed effectively with Zn(7)-MT for reactive nitrogen oxide species in reaction mixtures. Reaction of Cd(7)-MT with SNAP also required oxygen and light to react. In this case, only a fraction of the Cd(2+) bound to Cd(7)-MT was displaced by SNAP. Apo-metallothionein was much more reactive with SNAP and DEA-NO than Zn(7)- or Cd(7)-MT. TE671 and LLC-PK(1) cell lines were incubated with DEA/NO to examine the role that MT might play in the cellular reactions of this NO donor compound. Incubation of cells with 0-80 microM Zn(2+) for 24 h resulted in progressively increasing concentrations of Zn-unsaturated MT. One hour of cellular exposure to a range of DEA/NO concentrations followed by 24 h of incubation caused no evident acute toxicity at less than 0.45 mM. Preinduction of MT did not alter this response. The effects of DEA/NO on proteomic, metallothionein, and low molecular weight (LMW) thiol pools, including glutathione (GSH), were measured. Substantial fractions of the proteomic and LMW thiol pools underwent reaction with little dislocation of Zn(2+). In addition, one-third of the MT thiol pool reacted without labilizing any of the bound Zn(2+). These results demonstrated that it was free thiols associated with MT that reacted with DEA/NO not those bound to Zn(2+). Moreover, under the conditions of the experiments, DEA/NO reacted with the spectrum of cellular thiols in proportion to their fraction in the cytosol and did not preferentially react with MT sulfhydryl groups.