| dc.description.abstract | Cadmium (Cd) is a toxic heavy metal with no known physiological function in higher order animals. Previous studies in primary cultures of neonatal rat choroid plexus (CP) epithelial cells indicated Cd induced oxidative stress and stimulated apical choline transport, and suggested zinc (Zn) supplementation might abate both oxidative stress and modulation of transport. The objective of this thesis was to elucidate how Zn, a nutritive mineral normally accumulated by CP, attenuated oxidative stress. I hypothesize that Zn, which can function as a pro-antioxidant, abates Cd-induced oxidative stress either by induction of metallothionein-1 (MT-1) or enhancement of glutathione (GSH) biochemistry. Thus, in primary cultures of neonatal rat CP epithelial cells, I characterized the effects of sub-micromolar Cd and efficacy of Zn supplementation to attenuate Cd-induced cellular and oxidative stress without or with manipulation of GSH synthesis. To characterize the Cd-induced stress response, CP epithelial cells were treated with 0 or 500 nM CdCl_(2) in serum-free medium (SFM) for 12 h; samples were collected at 3, 6, 9, and 12 h. Induction of heme oxygenase-1 (HO-1), heat-shock protein 70 (HSP70), and metallothionein-1 (MT-1) in Cd-treated cells was compared to time-matched controls by immunoblot and qRT-PCR analyses. Cd induced the catalytic and
modifier subunits of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis. To elucidate the effects Zn supplementation in Cd-treated cells depleted of GSH, cells were supplemented for 48 h with 0 or 25 μM ZnCl_(2) alone or with 100 μM buthionine sulfoximine (BSO), an inhibitor of GCL, before treatment with 0 or 500 nM CdCl_(2) ± 100 μM BSO ± 10μM ZnCl_(2) in SFM for 12 h. By luminescence assay, intracellular GSH and oxidized glutathione (GSSG) concentrations were measured. Cd increased intracellular GSH and GSSG, but markedly decreased GSH:GSSG ratio. Inhibition of GSH synthesis exacerbated Cd-induced stress. However, Zn supplementation attenuated the stress response irrespective of BSO treatment, as per decreased induction of HSP70. These data indicate that CP adapts to low-dose Cd by up-regulation of stress proteins and GSH synthesis. Zinc supplementation also may attenuate Cd-induced cellular and oxidative stress, but cytoprotection is independent of GSH status. | en |
