Grantee Research Project Results
1998 Progress Report: Transport Mechanisms for Zinc Across Gill and Intestinal Epithelia of Fish
EPA Grant Number: R826104Title: Transport Mechanisms for Zinc Across Gill and Intestinal Epithelia of Fish
Investigators: Hogstrand, Christer
Institution: University of Kentucky
Current Institution: Kings College , University of Kentucky
EPA Project Officer: Hahn, Intaek
Project Period: November 15, 1997 through November 14, 2000
Project Period Covered by this Report: November 15, 1997 through November 14, 1998
Project Amount: $373,342
RFA: Exploratory Research - Environmental Biology (1997) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
To protect natural waters in a meaningful way, methods are being developed that allow us to predict the effects of a toxicant in each particular environment. Such methods should be based on an understanding of how toxicants interact with organisms and the physical environment. Unfortunately, there are still large gaps in our knowledge in these areas. For example, uptake mechanisms for toxic metals by animals are largely unresolved. The main focus of the current research is to delineate the pathways for zinc uptake in freshwater-acclimated rainbow trout and to characterize how zinc uptake is controlled. Our hypothesis is that there are regulated uptake pathways for zinc in fish that play important roles in toxicity and acclimation processes. Zinc was chosen as the focal substance of the project because our present knowledge about zinc uptake is farther advanced than that of most other toxic metals. Zinc also is one of the most problematic metals in terms of industries exceeding existing water quality criteria. Furthermore, zinc is added as a growth-enhancing nutritional supplement in the fish farming industry. This results in greater aquatic zinc concentrations due to poor uptake efficiency of this element. Finally, present data suggest that zinc is a useful model substance for the more toxic metal, cadmium.Progress Summary:
Branchial Zinc Transport. The ability of the rainbow trout gill to regulate zinc uptake was investigated during different forms of zinc treatments. Exposure to zinc via the water (23 µM) resulted in a substantial downregulation of zinc influx across the gills within 2 hours into the exposure. Likewise, waterborne zinc acutely depressed calcium influx. This is consistent with our previous data, which showed that apical entry of zinc and calcium into the gills occurs through the same transport mechanism. Interestingly, the response to intraperitoneal injection of zinc (50 µmol/kg) was a 225 percent increase in zinc influx while calcium influx remained unaffected. A possible interpretation of these results is that basolateral transfer of zinc is upregulated as a result of increased zinc levels in the system. The significance of such a mechanism would be to clear the primary site of acute zinc toxicity, the gills, from excess zinc. Indeed, in earlier studies we observed that the Jmax (maximum transport rate) for zinc influx is increased during waterborne zinc exposure, but the actual zinc uptake is reduced because of a dramatic decrease in zinc affinity. Thus, the overall compensatory response by the gills to elevated zinc in the environment may be a reduced apical entry and an increased basolateral efflux of zinc from the gill epithelium.We are using two distinct approaches to identify and characterize this apparently controlled mechanism to translocate zinc from the gill cells to the blood. Using isolated vesicles from the basolateral membrane of the gill epithelium we have provided substantial evidence that there exists a chloride-dependent zinc cotransport system that might be responsible for basolateral zinc efflux. The transporter is DIDS insensitive and transport is eliminated by addition of the zinc ionophore A23187. This efflux system also has proven to be markedly temperature sensitive, a hallmark of carrier-mediated transport. Using degenerate PCR primers to aligned conserved sequences of the mouse, rat, and gerbil ZnT-1 (a mammalian zinc-transporter), COT1 (a cobalt-transporter from yeast), and ZRC, (a yeast zinc resistance conferring gene), we have isolated and sequenced what appears to a segment of a fish ZnT-1 homologue. Our continued work should show whether or not the observed basolateral chloride-dependent zinc cotransport is mediated by ZnT-1 in rainbow trout gills.
Intestinal Zinc Transport. An in situ technique has been successfully developed to analyze the physiological characteristics of intestinal zinc uptake in live rainbow trout. Results from these experiments to date suggest two pathways of absorption, a saturable transporter-mediated pathway, and a diffusive pathway, that dominates at higher concentrations of zinc in the intestinal lumen. Influx and accumulation from the intestine into the body tissues appear to be linearly related to the intestinal zinc concentration, indicating a passive process involved in basolateral transport. As such, it is proposed that regulation of zinc uptake in the rainbow trout may be controlled at the apical membrane. Observations under further investigation suggest intestinal mucus plays a vital role in this process.
The influence of various compounds upon intestinal zinc uptake also has been investigated. Of those compounds analyzed to date, calcium has the greatest stimulatory effect upon zinc uptake. This stimulation appears to be at the brush border membrane of the epithelium. More mechanistic experiments are planned to clarify the nature of calcium-stimulated intestinal zinc absorption. The amino acids histidine and cysteine have specific effects upon intestinal zinc accumulation. L-histidine, but not D-histidine, promotes 65Zn accumulation in the systemic blood compartment of the fish. Despite this, both amino acids (i.e., histidine and cysteine), in a nonstereospecific manner, decrease zinc uptake. Glucose and the amino acid taurine had no significant effects on zinc transport. We are currently investigating pH dependence of zinc transport, and the specific role of thiol groups in zinc absorption.
Future Activities:
The research will progress largely as outlined in the original proposal. The most important change is that we have recently established a system to investigate interactions of waterborne zinc with the gills. This system is a cultured gill epithelium constructed from dispersed gill cells grown into a tissue on a semipermeable support. The cells produce a tight epithelium and orient uniformly, so that apical (i.e., facing water column) and basolateral (i.e., facing blood plasma) surfaces are maintained. A crucial feature is that we are able to stimulate differentiation of the epithelium into both respiratory cells (i.e., pavement cells) and ionocytes (i.e., chloride cells), as in the intact animal. The paramount significance to this improvement is that the ionocytes, which are believed to transport zinc and some other metals, can for the first time be studied in a controlled cell culture system.Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 27 publications | 7 publications in selected types | All 7 journal articles |
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Type | Citation | ||
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Burkhardt-Holm P, Bernet D, Hogstrand C. Increase of metallothionein-immunopositive chloride cells in the gills of brown trout and rainbow trout after exposure to sewage treatment plant effluents. Histochemical Journal 1999;31(6):339-346. |
R826104 (1998) R826104 (2000) R826104 (Final) |
not available |
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Dang ZC, Flik G, Ducouret B, Hogstrand C, Bonga SEW, Lock RAC. Effects of copper on cortisol receptor and metallothionein in the gills of rainbow trout Oncorhynchus mykiss. Aquatic Toxicology 2000;51(1):45-54. |
R826104 (1998) R826104 (2000) R826104 (Final) |
not available |
Supplemental Keywords:
heavy metal, Oncorhynchus mykiss, Zn, trace element, micronutrient, zinc transporter-1, biomembrane., RFA, Scientific Discipline, Toxics, Waste, Ecosystem Protection/Environmental Exposure & Risk, National Recommended Water Quality, Bioavailability, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, Environmental Microbiology, Zoology, Ecological Effects - Environmental Exposure & Risk, Biochemistry, Ecological Effects - Human Health, Ecology and Ecosystems, Ecological Indicators, cell transformation, transport contaminants, endocrine disrupting chemical, toxic metals, endocrine disruptors, Zinc, cellular mechanisms, aquatic ecosystems, amino acids, water quality, cadmium, fish , acclimation processes, rainbow troutRelevant Websites:
http://www.uky.edu/ArtsSciences/Biology/OIB/CHProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.