Kavitha Sankavaram

Zinc Status Alters Growth and Oxidative Stress Responses in Rat Hepatoma Cells

Zinc deficiency and excess influence cellular homeostasis and are believed to modulate apoptosis. Zinc also regulates cell growth and proliferation. Understanding of the role of zinc in the mechanisms associated with these changes is limited because of its diverse, complex, and cell-specific effects. Therefore, we investigated the oxidative stress responses and the underlying molecular mechanisms associated with the disruption of intracellular zinc homeostasis in H4IIE rat hepatoma cells. We found that zinc excess (100 μM) and DTPA (diethylenetriaminepentaacetic acid; 50–100 μM) induced zinc deficiency both generate reactive oxygen species (ROS) and decrease viability in H4IIE cells. However, cotreatment with the antioxidant, N-acetyl-L-cysteine (NAC) both reduced ROS production and protected cells from death. We additionally observed an increase in Bax mRNA and cytochrome c release from the mitochondria in DTPA-treated cells and an elevated expression of Fas/Fas ligand mRNA with zinc treatment. Both treatments increased p53 and MdM2 protein concentrations along with caspase 3/7 activity. These results suggest that zinc deficiency stimulates mitochondrial-dependent apoptosis whereas zinc activates the extrinsic-apoptotic pathway. Both decreasing and increasing cellular zinc concentrations modulate ROS mediated apoptosis and warrant further research on zinc mediated cancer chemoprevention in this and other cancer cell lines.

The effects of transformation and ZnT-1 silencing on zinc homeostasis in cultured cells.

We have previously demonstrated that reducing the availability of zinc with the extracellular chelator diethylenetriaminepentaacetic acid (DTPA) promotes efflux of (65)Zn from rat primary hepatocytes and pituitary cells, but increases retention of label in rat hepatoma (H4IIE) and anterior pituitary tumor (GH3) cell lines. To further understand this differential response between primary cells and the corresponding cancer cell lines, we investigated the effects of immortalizing primary cells on their zinc homeostasis. Rat primary hepatocytes were electroporated with the SV40 large T-antigen-coding plasmid pSV3-neo and selected for neomycin resistance. This resulted in cell division of the normally quiescent hepatocytes. When these cells were prelabeled with (65)Zn, DTPA decreased efflux of (65)Zn, similarly to hepatoma cells and differently from primary hepatocytes. This homeostatic change may be required to account for the greater zinc requirements of dividing cells and be mediated by alterations in the activity of zinc transporter ZnT-1, which is responsible for zinc efflux. To further understand the mechanism of DTPA-induced zinc retention, we down-regulated the expression of ZnT-1 in rat hepatoma cells using vector-based short hairpin RNA interference. Expression of ZnT-1 protein was reduced to approximately 50%. Down-regulation of ZnT-1 resulted in greater retention of (65)Zn in control cells. However, DTPA increased rather than decreased efflux of label from knockdown cells, suggesting that functional ZnT-1 is required for the decreased efflux in response to DTPA. We conclude that ZnT-1 expression is crucial for maintaining zinc homeostasis, in particular, for the enhanced retention of zinc in transformed cells when subjected to zinc deprivation.

Zinc retention differs between primary and transformed cells in response to zinc deprivation

Previous studies in our laboratory have demonstrated that reducing the availability of zinc with the extracellular metal chelator DTPA (diethylenetriaminepentaacetate) enhances, rather than inhibits, the thyroid hormone induction of growth hormone mRNA in GH3 rat anterior pituitary tumor cells. To understand the actions of the chelator on cellular zinc status, we observed the effects of DTPA on (65)Zn uptake and retention. DTPA reduced the uptake of (65)Zn by GH3 cells from the medium, but when GH3 cells were prelabeled with (65)Zn, it resulted in greater retention of the isotope. In primary hepatocytes, DTPA both reduced the uptake of (65)Zn from the medium and increased efflux from prelabeled cells. To investigate this difference, we studied the effects of DTPA on radioactive zinc flux in the H4IIE (rat hepatoma), MCF-7 (human breast cancer) and Hs578Bst (nontransformed human mammary) cell lines and in rat primary anterior pituitary cells. DTPA reduced the uptake of (65)Zn in all cell lines examined. DTPA increased the retention of (65)Zn in prelabeled H4IIE, MCF-7 and Hs578Bst cells but reduced it in primary pituitary cells. Time course experiments showed that (65)Zn efflux is shut down rapidly by DTPA in transformed cells, whereas the chelator causes greater efflux from primary hepatocytes over the first 6 h. Experiments with (14)C-labeled DTPA confirmed that this chelator does not cross cell membranes, showing that it operates entirely within the medium. Expression of ZnT-1, the efflux transporter, was not affected by DTPA in H4IIE cells. Thus, zinc deprivation enhanced zinc retention in established cell lines but increased efflux from primary cells, perhaps reflecting differing requirements for this mineral.

Zinc: Physiology, Dietary Sources, and Requirements

Zinc is a relatively abundant trace element that performs a wide variety of functions in the body. Because of its size and charge characteristics, it is used by thousands of proteins to stabilize structure and assist function. These proteins are largely enzymes and transcription factors that are involved in many bodily processes, including growth and immune function. Zinc is also widely distributed in foods, though bioavailability may be limited in plant foods by phytate. Absence of a good method for assessing status makes estimation of prevalence of deficiency problematic, but mild deficiencies may be common.

Rapid homeostatic response of H4IIE cells to diethylenetriaminepentaacetic acid is not due to changes in the amount or localization of ZnT-1 protein

We have demonstrated that reducing zinc availability with the extracellular chelator diethylenetriaminepentaacetic acid (DTPA) causes rapid inhibition of cellular zinc efflux in H4IIE hepatoma cells but increases zinc efflux in primary hepatocytes. Similar differences were also observed between the rat anterior pituitary cell line GH3 and primary anterior pituitary cells. We hypothesized that the difference between the transformed and primary cells is due to differential regulation of ZnT-1 or SLC-30A-1 because this is the only zinc efflux transporter localized to the plasma membrane. The effects of DTPA (50 μM) and zinc (100 μM) treatment on messenger RNA (mRNA) and protein expression and protein localization of ZnT-1 were studied in H4IIE cells and primary hepatocytes. Although zinc tended to increase ZnT-1 mRNA in H4IIE cells, DTPA had no effect on ZnT-1 mRNA and protein expression in either hepatoma cells or hepatocytes. Although ZnT-1 is thought to be localized on the plasma membrane, this localization was not seen in these liver cells where ZnT-1 was distributed throughout the cytoplasm. Vesicular localization of ZnT-1 appeared to increase with zinc treatment. Total zinc content was reduced by DTPA in H4IIE cells. Diethylenetriaminepentaacetic acid also reduced metallothionein 1 mRNA, reflecting this reduction in cellular zinc. We conclude that the rapid homeostatic response of cells to altered zinc availability must be attributed to a transporter other than ZnT-1 or to changes in the activity of ZnT-1 by a novel mechanism.