Takahiro Yanagiya

The role of zinc transporters in cadmium and manganese transport in mammalian cells

To understand the mechanism of cadmium accumulation, it is important to know the precise mechanisms of transport systems for other metals. Recently, utilization of genomics and metallomics has clarified the involvement of specific metal transporter(s) in cadmium uptake. Studies with metallothionein (MT)-null cadmium-resistant cells have revealed the involvement of the manganese/zinc transport system in cadmium uptake. Genomic studies of strain differences in sensitivity to cadmium-induced testicular hemorrhage revealed that a zinc transporter, Zrt-, Irt-related protein (ZIP) 8 encoded by slc39a8, is responsible for the strain difference. Ectopic expression of ZIP8 in various cells enhanced the uptake of cadmium, manganese, and zinc. ZIP8-transgenic mice showed high expression of ZIP8 in the vasculature of testis and apical membrane of proximal tubules in kidney, and exhibited enhanced cadmium accumulation and toxicity when treated with cadmium. The expression of ZIP8 was found to be down-regulated in MT-null cadmium-resistant cells, in which the uptake rates of both cadmium and manganese were decreased. These data suggest that ZIP8 plays an important role in the uptake of both cadmium and manganese in mammalian cells. The role of ZIP14 in the uptake of cadmium and manganese is also discussed.

Simian virus 40-transformed metallothionein null cells showed increased sensitivity to cadmium but not to zinc, copper, mercury or nickel.

Primary cultured embryonic cells derived from mice with disrupted metallothionein (MT) I and II genes and from control mice were transformed with a plasmid encoding the simian virus 40 (SV40) large T antigen. The resulting MT-/- and MT+/+ cell strains showed similar cell morphology, cell cycle and no significant differences in glutathione levels or in the activities of glutathione-related enzymes and antioxidant enzymes. The MT-/- cells were more sensitive to Cd than MT+/+ cells, though no increase in the sensitivity to Zn, Cu, Hg or Ni were observed in MT-/- cells. MT+/+ cells accumulated more Cd than MT-/- cells but showed less lesion, suggesting the role of MT induced by Cd in MT+/+ cells as a scavenger of toxic Cd ion. These results suggest a dominant protective role of MT against Cd compared with other metals. SV40-transformed MT-/- cells seem to be a useful tool for the investigation of cellular function of MT.

Reduced uptake and enhanced release of cadmium in cadmium-resistant metallothionein null fibroblasts.

Metallothionein (MT) is known to play a predominant role in the protection of cells from cadmium (Cd) toxicity. To investigate other factors involved in Cd resistance, we established Cd-resistant cell lines from simian virus 40-transformed MT null fibroblasts. Cd-resistant MT null cells, Cd-rA7 and Cd-rB5, developed approximately 10-fold resistance to Cd compared to parental cells, but showed no cross-resistance to Zn, Cu, Hg, Ni, As, cisplatin or H2O2. Accumulation of Cd in the resistant cells was 13-18% of that of parental cells after treatment with Cd for 24 h. A short-term experiment revealed that the rate of Cd incorporation into the Cd-resistant cells was suppressed, and the rate of Cd release was enhanced in the resistant cells compared with that of parental cells. These results indicate that the altered transport of Cd, slow uptake and rapid release, may confer resistance to Cd on the Cd-resistant cells established from MT null fibroblasts.

Cross-resistance of cadmium-resistant cells to manganese is associated with reduced accumulation of both cadmium and manganese.

The mechanism of cellular entry of cadmium remains unclear. We have previously established cadmium-resistant cells from mouse embryonic cells of metallothionein (MT)-null mice, and demonstrated that the down-regulation of a zinc transporter, Zrt/Irt-related protein (ZIP) 8, was responsible for the reduced cadmium incorporation into cells. In the present study, we developed cadmium-resistant cells (A+70 and B+70) from mouse embryonic cells of MT-expressing wild-type mice. The LC₅₀ values of CdCl₂ for A+70 and B+70 cells were about 200 μM while that of the parental cells was 30 μM. We found that the cadmium resistance of these cells was conferred not only by enhanced expression of MT, but also by a decrease in cadmium accumulation. Since the uptake rates of cadmium into A+70 and B+70 cells were lowered, we determined the expression levels of the metal transporters and channels potentially involved in the cellular uptake of cadmium. We found a down-regulation of multiple transport systems, including ZIP8, divalent metal transporter 1 (DMT1), and α₁ subunits of L-type (Ca(V)1.2) and T-type (Ca(V)3.1) voltage-dependent calcium channels, in A+70 and B+70 cells. Furthermore, A+70 and B+70 cells exhibited cross-resistance to cytotoxicity of MnCl₂, probably due to a marked decrease in manganese uptake in these cells. These results suggest that the suppressed expression of ZIP8 and DMT1, which are known to have affinities for both cadmium and manganese, may be responsible for the reduction in the uptake, and consequently the cytotoxicity, of cadmium and manganese in A+70 and B+70 cells.

Induction of renal metallothionein synthesis bycis-diamminedichloroplatinum (II) in glutathione-depleted mice

Effects of glutathione (GSH) depletion on nephrotoxicity of cis-diamminedichloroplatinum(II) (cis-DDP) and induction of renal metallothionein (MT) synthesis elicited by cis-DDP treatment has been studied using mice. Pretreatment with L-buthionine-SR-sulfoximine (BSO) reduced the GSH level in the kidneys to 20% of the control level at the time of cis-DDP administration. Administration of 45 μmol/kg cis-DDP did not change the blood urea nitrogen (BUN) level, determined as an indicator of nephrotoxicity, until 96 h after administration, whereas BSO pretreatment raised the BUN level as early as 48 h after cis-DDP (45 μmol/kg) treatment. The level of renal MT was also increased in the GSH-depleted mice and was changed in parallel with the BUN level after cis-DDP treatment. Level of plasma fibrinogen, one of acute phase proteins, increased by cis-DDP in GSH-depleted mice. The increase was reduced by dexamethasone pretreatment, although the renal MT level increased by BSO and cis-DDP was not changed by dexamethasone pretreatment. These results suggest that the induction of renal MT synthesis by cis-DDP in BSO-pretreated mice was not related with the acute phase responses such as cytokines induction. Although the mechanism of the induction of MT synthesis by cis-DDP in GSH-depleted mice is unclear, it is possible that MT is induced as an antidote against oxidative stress, which may be a major cause for nephrotoxicity of cis-DDP enhanced by GSH depletion.