UBC Theses and Dissertations
Effect of zinc nutriture on growth and the labile intracellular pool of zinc in 3T3 cells and historically reactive zinc in rats Paski, Shirley Catherine
Zinc is an essential nutrient that participates in and regulates many activities that are instrumental to growth including DNA, RNA and protein synthesis. A diet deficient in zinc has severe physiological consequences, including suppressed growth, yet total tissue zinc levels appear unaffected. From this observation, it appears that small, subtle changes in the quantity or availability of intracellular zinc may regulate certain biochemical processes, including growth. Most zinc in biological systems is tightly bound to proteins or nucleic acids. However, a small, labile intracellular pool of zinc (LIPZ) exists that is either in free form or loosely bound to protein. The LIPZ may be identified using fluorescent probes, like N-6-methoxy-paratoluenesulphonamide (TSQ). The purpose of this research was to study the effects of zinc nutriture on modulating the LIPZ size and to examine the relationship between the LIPZ size and growth, in vivo and in vitro. Experiment 1 was designed: 1) to characterize the relationship between zinc nutriture and the LIPZ size under varying zinc nutriture in 3T3 cells, and 2) to study the effects of modulating LIPZ size via varying zinc nutriture on growth in 3T3 cells. Quiescent cells were exposed to media containing varying zinc nutriture [DMEM (control); Chelex-treated, low zinc DMEM (CDMEM), or CDMEM replenished with 5, 10, 20 or 40µM ZnSO4 (CDMEM+5, 10, 20, or 40)] for 24 or 48 hours. Increasing zinc nutriture resulted in increased LIPZ size after both 24 and 48 hours of exposure to treatment media. Furthermore, the decrease in LIPZ size that occurred with exposure to CDMEM media was accompanied by a decrease in cell proliferation and overall growth that could be replenished by increasing zinc nutriture. Experiment 2 was designed: 1) to characterize the relationship between dietary zinc and the tissue histochemically reactive zinc in rats, and 2) to study the effects of modulating histochemically reactive zinc via varying dietary zinc intake on growth in rats. 21-day-old male Wister rats were fed treatment diets containing either low (LZ, 3mg Zn/kg diet), control (CZ, 31mg Zn/kg diet) or high (HZ, 155mg Zn/kg diet) levels of zinc ad libitum. A pair-fed control (PF, 31mg Zn/kg diet) diet was included that was fed the CZ diet in the amount that the LZ rats consumed the previous day. After 14 or 42 days on the treatment diets, rats were killed and muscle, liver, and small intestine samples were collected. The rats that were fed the LZ diet for 14 days had a decreased histochemically reactive zinc and cell proliferation in small intestine compared to the pair-fed controls. Neither the histochemically reactive zinc nor cell proliferation appeared to be affected by zinc nutriture in any of the other tissues samples. Although histochemically reactive zinc and cell proliferation appeared to be largely unaffected by zinc nutriture, total tissue zinc in liver and intestine in addition to overall growth was reduced in rats fed LZ diets compared to PF controls. Experiment 3 was designed to investigate the interaction between cell proliferation rate and the LIPZ size under varying zinc nutriture in 3T3 cells. Quiescent cells were exposed to media containing varying zinc nutriture (control, CDMEM, or CDMEM+20), with or without growth factors (EGF, PDGF, and IGF-1), for 24 or 48 hours. Using EGF, PDGF, and IGF-1 to increase the rate of cell proliferation and overall growth increased the LIPZ size in 3T3 cells. Cells grown in CDMEM+20 experience the greatest increase in LIPZ size upon growth factor addition. In conclusion, there appears to be a relationship between zinc nutriture, the LIPZ size, and cell proliferation in 3T3 cells, but not in vivo. This apparent discrepancy may be due to differences in tissue zinc availability, exposure time, tissue preparation method, and/or intracellular zinc storage and distribution mechanisms.
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