Introduction

1. Point A to C: Copper and Cadmium Transport in Caco-2 Cells After DMT-1 Knockdown Dale Hatrock, York College of Pennsylvania Biology Department http://periodictable.com/ http://periodictable.com/ • Introduction • Cadmium (Cd) is a toxic transition metal with LC50 of 229.9 mg/m3 for 4 hours of exposure. It forms 2+ ions. • Copper(Cu) is a nutritive transition metal contained in the human body at levels of 1.4 to 2.1 mg/kg. It forms both 1+ and 2+ ions. • Caco-2 is a human corectal cancer cell line that specializes in tissue culture to resemble intestinal cells. • DMT-1 (also known as natural resistance-associated macrophage protein 2, Nramp-2) is a divalent metal transporter with high affinity for iron, cadmium, and debated affinity for copper. It is expressed in nearly every cell type. It is very highly expressed in the small intestine and kidney, and is associated with intake of both nutritive and toxic metals. • Figure 1:Uptake rates of various metals in Xenopusoocytes expressing DMT-1. Oocytes were injected with human DMT-1 RNA. Figure from Illing et al., 2012. • Hypotheses/Objectives • Identify how available levels of copper impacts uptake of cadmium. • Characterize how copper and cadmium influence DMT-1 expression. • Determine if DMT-1 is implicated in cadmium and copper uptake. • Review of Literature • Diets deficient in iron can lead to increased levels of DMT-1 expression upon exposure to cadmium, resulting in increased cadmium in body mass (Ryu et al., 2004). • DMT-1’s ability to transport copper is still debated, with Jiang and Collins (2012) stating that DMT-1 can transport copper under low-iron conditions, while Arredondo et al. (2003) state that DMT-1 is a physiologically relevant Cu1+ transporter. • Illing et al., (2013) does not support that DMT-1 is a relevant copper transporter. • Figure 3: Efficacy of knockdown construct. Levels of DMT-1 and GAPDH mRNA as examined by Northern Blot. Figure from Bannon et al. (2003). • Expected Results • Wild type cells exposed to copper only will uptake the copper, while cells exposed to both copper and cadmium will uptake the cadmium preferentially. • DMT-1 protein expression will increase at an mRNA level for 15 minutes following the introduction of the uptake buffer. Expressed levels of DMT-1 will increase from 5 minutes to 15 minutes after introduction of buffer, at which point the needed protein level will be expressed. • Caco-2 cells are the standard model of human intestinal cells, and are a suitable host for transfection (Sambury et al., 2005). • Stable DMT-1 knockdown cell lines have been created (Bannon, et al., 2003). • Figure 2: Knockdown construct used by Bannon et al., 2003. This construct displayed a significant (p < 0.01) reduction in DMT-1 expression due to interference with mRNA production. Figure from Bannon et al. (2003). • Presence of cadmium will decrease levels of copper uptake. Copper uptake will not be as impacted by the knockout of DMT-1 as cadmium uptake, suggesting an alternate route of uptake for copper, with DMT-1 being a peripheral transport protein. • Figure 4: Gel stain showing protein expression trends. After exposure to uptake buffer, DMT-1 levels begin to increase, reaching maximum expression 15m after exposure. • Figure 5: Uptake of Cu2+ and Cd2+ by both wild type and knockdown cell lines. Cd2+ is transported by wild type cells via DMT-1, with little inhibition by Cu2+. Cu2+ uptake is strongly inhibited the presence of Cd2+, suggesting that DMT-1 is a secondary transport protein of Cu2+. Cd2+ uptake is strongly inhibited by the knockdown of DMT-1, which is consistent with findings that DMT-1 is an important protein in the uptake of Cd2+. Cu2+ uptake is less impacted by the knockdown, suggesting an additional route of Cu2+ uptake. • Works Cited • Bannon, D.I., Abounader, R., Lees, P.S.J., Bressler, J.P., 2003 Effect of DMT1 Knockdown on iron, cadmium and lead uptake in caco-2 cells. American Journal of Physiology – Cell Physiology. 284:44-50. • Anthony C. Illing, A.C., Shawki, A., Cunningham, C.L., Mackenzie, B. 2012. Substrate Profile and Metal-ion Selectivity of Human Divalent Metal-ion Transporter-1. Journal of Biological Chemistry 287:30485-30496 • Sambuy, Y., De Angelis I., Ranaldi,G., Scarino, M.L., Stammati, A. , Zucco, F. 2005. 2005 The Caco-2 Cell Line as a model of the intestinal barrier: Influence of cell and culture-related factors on Caco-2 cell Functional characteristics. Cell Biology and Toxicology. 21: 1-26. • Ryu, D.Y., Lee, S.J., Park, D.W., Choi, B.S., Klaassen, C.D., Park, J.D. 2004. Dietary iron regulates intestinal cadmium absorption through iron transporters in rats. Toxicology Letters, 152(1):19-25. • Jiang, L., Garrick, M.D., Garrick, L.M., Zhao, L., Collins., J.F. 2013. Divalent metal transporter 1 (DMT1) Mediates Copper Transport in the Duodenum of Iron-Deficient Rats and When Overexpressed in Iron-Deprived HEK-293 Cells. Journal of Nutrition. • Arredondo, M., Munoz, P., Mura, C.V., Nunez, M.T. 2003. DMT1, a physiologically relevant apical Cu1+ transporter of intestinal cells. American Journal of Cell Physiology. 284(6):1525-1530. • Acknowledgements • I would like to thank Dr. Ronald Kaltreider for his guidance and advice. METHODS Caco-2 cells in tissue culture Expose cells to uptake buffer for 30 min. Expose cells to uptake buffer for 30 min. Transfect Caco-2 cells to knockdown DMT-1 expression Wash cells, lyse cells at 5 minute intervals following exposure. Expose cells to uptake buffer for 30 min. Wash cells, lyse cells. Wash cells, lyse cells. Assay cell lysate for DMT-1 protein and mRNA level via Western and Northern blot Analyze cell lysate for Cd and Cu levels using ICP. Analyze cell lysate for Cd and Cu levels using ICP.