Zinc, Zinc Transporters, and Cadmium Cytotoxicity in a Cell Culture Model of Human Urothelium

Abstract

We explored the potential role of zinc (Zn) and zinc transporters in protection against cytotoxicity of cadmium (Cd) in a cell culture model of human urothelium, named UROtsa. We used real-time qRT-PCR to quantify transcript levels of 19 Zn transporters of the Zrt-/Irt-like protein (ZIP) and ZnT gene families that were expressed in UROtsa cells and were altered by Cd exposure. Cd as low as 0.1 µM induced expression of ZnT1, known to mediate efflux of Zn and Cd. Loss of cell viability by 57% was seen 24 h after exposure to 2.5 µM Cd. Exposure to 2.5 µM Cd together with 10-50 µM Zn prevented loss of cell viability by 66%. Pretreatment of the UROtsa cells with an inhibitor of glutathione biosynthesis (buthionine sulfoximine) diminished ZnT1 induction by Cd with a resultant increase in sensitivity to Cd cytotoxicity. Conversely, pretreatment of UROtsa cells with an inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (aza-dC) did not change the extent of ZnT1 induction by Cd. The induced expression of ZnT1 that remained impervious in cells treated with aza-dC coincided with resistance to Cd cytotoxicity. Therefore, expression of ZnT1 efflux transporter and Cd toxicity in UROtsa cells could be modulated, in part, by DNA methylation and glutathione biosynthesis. Induced expression of ZnT1 may be a viable mechanistic approach to mitigating cytotoxicity of Cd.

Keywords: BSO; DNA methylation; ZIP zinc transporters; ZIP14; ZnT zinc transporters; ZnT1; aza-dC; cadmium; glutathione; qRT-PCR; urothelium.

Figure 1

Time-course and dose-response analyses for an induced expression of the ZnT1 zinc transporter gene by cadmium. (A) Expression levels of the ZnT1 gene as a function of exposure durations and Cd²⁺ concentrations; (B) expression levels of the ZnT1 gene 24 h and 48 h after exposure to varying Cd²⁺ concentrations. Fold change or a ratio was defined as the number of transcripts of a given gene relative to β-actin in control cells divided by number of transcripts of the same gene in treated cells relative to β-actin. ZnT1 expression in (A,B) were from different batches of cells. An effect of 48-h exposure was not assessed for 0.1 and 1.5 µM Cd concentrations.

Figure 2

Effects of exposure to cadmium alone or cadmium plus zinc on cell viability and expression levels of ZIP and ZnT zinc transporter genes. (A) Change in cell viability following exposure to only cadmium; (B) zinc protection against cadmium-induced loss of cell viability; (C) expression levels of ZnT1 gene in cells exposed to cadmium and zinc; (D) expression levels of ZnT5, ZnT7, ZIP8, and ZIP14 genes in cells exposed to cadmium and zinc. Fold change or a ratio was defined as number of transcripts of a given gene relative to β-actin in control cells divided by number of transcripts of the same gene in treated cells relative to β-actin. Cell viability data in (A,B) were assessed with different batches of cells. Expression of ZnT1 in (C) and expression of other ZnT and ZIP transporters in (D) were assessed with an identical cell batch.

Figure 2

Effects of exposure to cadmium alone or cadmium plus zinc on cell viability and expression levels of ZIP and ZnT zinc transporter genes. (A) Change in cell viability following exposure to only cadmium; (B) zinc protection against cadmium-induced loss of cell viability; (C) expression levels of ZnT1 gene in cells exposed to cadmium and zinc; (D) expression levels of ZnT5, ZnT7, ZIP8, and ZIP14 genes in cells exposed to cadmium and zinc. Fold change or a ratio was defined as number of transcripts of a given gene relative to β-actin in control cells divided by number of transcripts of the same gene in treated cells relative to β-actin. Cell viability data in (A,B) were assessed with different batches of cells. Expression of ZnT1 in (C) and expression of other ZnT and ZIP transporters in (D) were assessed with an identical cell batch.

Figure 3

Effects of cadmium in UROtsa cells pretreated with BSO or aza-dC. (A) An analysis of cell viability reduction; (B) changes in ZnT1 expression in BSO-treated UROtsa cells; (C) an analysis of resistance to loss of cell viability; (D) changes in ZnT1 expression in aza-dC-treated UROtsa cells. Fold change or a ratio was defined as number of transcripts of a given gene relative to β-actin in control cells divided by number of transcripts of the same gene in treated cells relative to β-actin. Cell viability in (A) and ZnT1 expression in (B) were assessed with an identical cell batch. Cell viability in (C) and ZnT1 expression in (D) were assessed with an identical cell batch.

Figure 4

Effects of cadmium on expression of selected zinc transporters and viability of BSO- and aza-dC-treated cells. (A) Changes in expression levels of ZnT5, ZnT7, ZIP8, and ZIP14 in BSO-treated cells caused by 1 µM Cd²⁺; (B) changes in expression levels of ZnT5, ZnT7, ZIP8, and ZIP14 in aza-dC-treated cells caused by 2.5 µM Cd²⁺; (C) changes in expression levels of ZnT5, ZnT7, ZIP8, and ZIP14 in aza-dC-treated cells caused by 5 µM Cd²⁺. Effects of 24, 48, and 72 h of cadmium exposure on viability of UROTsa cells pretreated with 2.5 µM aza-dC (D). Fold change or a ratio was defined as number of transcripts of a given gene relative to β-actin in control cells divided by number of transcripts of the same gene in treated cells relative to β-actin. Expression profiles in (A) were based on RNA samples from cells in Figure 3B. Expression profiles in (B,C) were based on RNA samples from cells in Figure 3D. An effect of aza-dC treatment duration in (D) was assessed with a different batch of cells.