Figure 6.

Effects of Zinc supplementation and inhibition of glutathione synthesis by l-buthionine sulfoximine (BSO) on gene expression for rat heat shock protein-70 (Hspa4), heme oxygenase (Hmox1), metallothionein (Mt1), GCLC (Gclc), and GCLM (Gclm) expression in choroid plexus epithelial cells (CPECs) exposed for 12 h to 500 nM CdCl₂. CPECs were first incubated (48 h) in maintenance medium without Zinc; i.e., non-supplemented cells, or with 25 µM ZnCl₂; i.e., Zn-supplemented cells. (A) A representative group of non-supplemented CPECs was treated in serum-free medium (SFM) for 12 h, but not subjected to further treatment; in parallel, representative Zn-supplemented cells were treated in SFM with 10 µM ZnCl₂ for 12 h, but not subjected to further treatment. (B,C) In the absence of BSO (--No BSO--), a group of non-supplemented cells was pre-treated (12 h) in SFM and then divided into two sets; one set was incubated (12 h) in SFM without cadmium (No Cd/No Zn), while the other was exposed (12 h) to 500 nM CdCl₂ in SFM (Cd). In absence of BSO as well (--No BSO--), a group of Zn-supplemented cells was pre-treated (12 h) in SFM with 10 µM ZnCl₂ and then divided into two sets: one set was incubated (12 h) in SFM with 10 µM ZnCl₂ (Zn), while the other was exposed (12 h) to 500 nM CdCl₂ in SFM with 10 µM ZnCl₂ (Zn + Cd). To test the effects of inhibition of glutathione synthesis, BSO was added to the SFM used for pre-treatment and CdCl₂ exposure of non-supplemented and Zn-supplemented CPECs. In the presence of BSO (--BSO--), a group of non-supplemented cells was pre-treated (12 h) in SFM with 100 µM BSO and divided into two sets: one was incubated (12 h) in SFM with 100 µM BSO (BSO, No Cd/No Zn), while the other set was exposed (12 h) to 500 nM CdCl₂ in SFM with 100 µM BSO (BSO + Cd). In the presence of BSO as well, (--BSO--), a group of Zn-supplemented cells was pre-treated (12 h) in SFM with 100 µM BSO and 10 µM ZnCl₂ and then divided into two sets: one was incubated (12 h) with 100 µM BSO and 10 µM ZnCl₂ (BSO + Zn); the other was exposed (12 h) to 500 nM CdCl₂ in SFM 100 µM BSO and 10 µM ZnCl₂ (BSO + Zn + Cd). All treatments were performed at 37 °C. The mRNA expression was analyzed in triplicate by quantitative real-time polymerase chain reaction (qRT-PCR); mRNA expression for each test gene was normalized to mRNA for rat β-actin (Actb) and GAPDH (Gapdh). Fold-induction was calculated as the ratio of normalized gene expression in Cd-exposed cells to that in time-matched controls. The mRNA was analyzed in triplicate in five separate culture preparations. Data are presented as means ± SE; n = 5; * p < 0.05 vs. Control; § p < 0.05 vs. Cd; # p < 0.05 Zn + BSO + Cd vs. BSO + Cd.