The overall goal of the present research project was to investigate the physiology of dietary iron absorption and its interactions with the uptake and metabolism of other divalent metals, especially cadmium, in freshwater fish, using rainbow trout (Oncorhynchus mykiss) as a model species. Using intestinal sac preparations, iron absorption was found to occur along the entire intestinal tract of fish, with anterior intestine being the major site of absorption compared to either mid or posterior intestine. Ferrous iron was more bioavailable than ferric iron, and the uptake of ferrous iron was significantly reduced at alkaline pH (p < 0.05). These findings suggested that a homolog of the mammalian apical ferrous iron transporter, divalent metal transporter-1 (DMT1, a Fe2+/H+ symporter), is involved in the absorption of iron in the fish intestine. Ferric iron appeared to be absorbed through the same pathway as ferrous iron following reduction by an apical ferric reductase. Several divalent metals, both essential (nickel, copper and zinc) and non-essential (cadmium and lead), inhibited intestinal ferrous iron absorption in fish. Importantly, elevated luminal iron reciprocally reduced the accumulation of cadmium in the fish intestine, indicating the significance of the iron transport pathway in dietary cadmium absorption. Two different DMT1 isoforms, Nramp-â and -ã, were found to be expressed along the entire gastrointestinal tract of fish. My study showed that in isolated rainbow trout enterocytes, ferrous iron uptake occurred through a saturable and proton-dependent process, providing further evidence of DMT1-mediated ferrous iron transport. Both cadmium and lead inhibited ferrous iron uptake in the enterocytes in a concentration-dependent manner. Kinetic characterization revealed that the apparent affinity for ferrous iron uptake is significantly decreased (increased Km) in the presence of either cadmium or lead (p < 0.05), whereas the maximum uptake rate (Jmax) remains unchanged. These results indicated that the interaction between ferrous iron and cadmium or lead is competitive in nature, and the uptake of these metals occurs through a common transport pathway (likely DMT1). The uptake characteristics of cadmium were further examined in isolated rainbow trout enterocytes, and my findings indicated that in addition to DMT1, cadmium uptake can be mediated by zinc transport pathway (ZIP8, a Zn2+/HCO3- symporter). My study also showed that cysteine-conjugated cadmium was readily bioavailable to fish enterocytes, possibly via a cysteine-specific transport pathway. The efflux of cadmium from the enterocytes was found to occur via an ATPase-driven pathway. On the other hand, chronic exposure to dietary cadmium at an environmentally-relevant concentration significantly increased cadmium burden in target organs as well as in the whole-body of fish (p < 0.05). Exposure to dietary cadmium increased the mRNA expression level of key stress-inducible proteins such as metallothioneins (MT-A and MT-B) and heat shock proteins-70 (HSP-70a and HSP-70b). Interestingly, each MT and HSP-70 mRNA isoform responded differently in various target organs of fish following dietary cadmium exposure. Fish exposed to dietary cadmium also exhibited an increase in the hepatic transferrin mRNA level as well as the plasma transferrin protein level, indicating the role of transferrin in cadmium handling in fish. Importantly, an iron-supplemented diet reduced cadmium burden in the gut and the whole-body, and ameliorated the expression of MT and HSP-70 genes in fish. These results suggested the protective effects of elevated dietary iron against chronic dietary cadmium toxicity in fish. Overall, findings from the present research project provided novel and important physiological and molecular insights into the uptake, interactions and homeostasis of dietary divalent metals in freshwater fish. This information greatly enhances our current understanding of the toxicological implications for dietary metal exposure in metal contaminated wild fish populations, and may ultimately help the regulators to develop better strategies for ecological risk assessment of metals.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-06142011-112636 |
| Date | 11 July 2011 |
| Creators | Kwong, Wai Man (Raymond) |
| Contributors | Niyogi, Som, Blakley, Barry, Van Kessel, Andrew, Janz, David, Wei, Yangdou, Wilkie, Michael |
| Publisher | University of Saskatchewan |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-06142011-112636/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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