Grantee Research Project Results
2000 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: 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, 1999 through November 14, 2000
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, there is a need to develop methods by which we can predict the effects of a toxicant in each particular environment. Such methods must 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 objective of the project has been to delineate the pathways of Zn uptake in freshwater trout, and to characterize how Zn uptake is controlled. We hypothesized that there are regulated uptake pathways for Zn in fish that play important roles in toxicity and acclimation processes. Zn was chosen as the focal substance of the project because our present knowledge about Zn uptake is farther advanced than that of most other toxic metals. Furthermore, present data suggest that Zn is a useful model substance for the more toxic metal, cadmium.Progress Summary:
Research during the reporting period provided evidence that waterborne zinc and copper exposure induces production of the metal detoxifying protein, metallothionein (MT) in the gills of fish in a cell-specific manner. The ion transporting chloride cells predominantly express MT, supporting other evidence that this is the site of branchial copper and zinc uptake and that MT is induced as a defense mechanism in these cells. Interestingly, MT expression was observed exclusively in chloride cells of brown trout exposed in situ to a zinc contaminated sewage effluent in Switzerland. This linked effects observed in the laboratory to a response during environmental exposure to zinc. We showed earlier that zinc enters the gill epithelium apically through a transporter that is shared with calcium. Recent studies, included in the reporting period, have provided new evidence that zinc transport across the basolateral membrane of the gill epithelium is mediated by a DIDS insensitive chloride, zinc co-transport mechanism. In ongoing studies, we have taken a functional genomics approach to study gill zinc transport. Through previous work, we know that transporter properties for zinc change during exposure. Through hybridization of Fugu gill cDNA arrays with probes derived from poly A+ RNA from zinc exposed and control fish, respectively, we have identified a large number of genes that were differentially expressed in response to waterborne zinc. These genes now are being sequenced and identified. We hope to identify novel genes involved in regulation of zinc uptake and defense against waterborne zinc exposure through this procedure. Screening Fugu cDNA arrays with a probe derived from a partial sequence of the mouse ZnT-1 gene, we have isolated and sequenced the first fish zinc transporter, fuZnT-1. We currently are investigating the presence and expression of ZnT-1 in other fish species.Although uptake of Zn across the gill epithelium is of primary concern for acute Zn toxicity, intestinal Zn uptake is likely more important for the whole body Zn accumulation. Movement of Zn and other metals across the intestinal epithelium has been difficult to study. Thus, as a section of this project, we have used a newly developed cannulation technique to produce new data on intestinal Zn uptake in freshwater fish. We have characterized the physiological properties of the intestinal Zn uptake and investigated the modulating effects of inorganic ions, amino acids, and peptides on Zn uptake.
The initial characterization of zinc absorption in cannulated trout has demonstrated that only a saturable component of uptake exists. This differs from the proposed situation in mammals and in the winter flounder, a marine teleost. Whether these differences are a consequence of technique or due to environment- or species-specific effects remains to be proven. However, there is a strong suggestion that the in vivo nature of the preparation has an important influence. Massive intestinal mucus secretion was described upon exposure to luminal zinc. If the linear, non-saturable uptake component described by other authors is a diffusively driven process, then it may be an artifact of various in vitro or less physiological condition. If the natural response is to secrete mucus this may occlude diffusive flow preventing diffusive zinc uptake. Data describing unaltered intestinal tissue water content supports this notion. It is possible that the mucus secretion in response to luminal zinc also could act to block the absorption of nutrients such as sugars and amino acids, causing secondary deleterious effects. This suggests that pollutants need not be absorbed to cause harm.
It is hypothesized that mucus may have important functions as a barrier to and also as a promoter of zinc absorption. Mucus traps up to 74 percent of perfused zinc. Accompanied by the continual sloughing of both mucus and epithelial cells, the majority of perfused zinc is excluded from absorption. However, at low, nutritionally relevant zinc levels, it appears that mucus facilitates the uptake of zinc. Thus, the mucus seems to function in essence as a buffer for zinc uptake, stimulating at lower luminal zinc concentrations and inhibiting at higher concentrations.
Evidence also suggests that the levels of intracellular zinc may be regulated by basolateral export, and there is a possibility that a potassium-mediated efflux mechanism is involved. The use of a thiol group reagent has further determined that apical uptake and passage into the intestine does not depend upon functional sulphydryl groups, but the movement of zinc into the circulation is highly thiol-dependent.
Metals were discovered to have specific effects upon the pattern of zinc accumulation. Cadmium stimulated additional mucus secretion and caused a decrease in intestinal zinc accumulation without an effect upon appearance of zinc in the circulation and rest of the body. Conversely, both copper and calcium decreased the zinc-induced mucosecretory response; however, the same amount of zinc bound to the mucus/epithelial tissue, lead to an increase in epithelial zinc concentration. These elements had no effect upon intestinal zinc accumulation, but decreased the passage of zinc into the post-intestinal tissues. The effect on tissue distribution appeared independent of changes at the mucus barrier, as magnesium also decreased mucus secretion but was without effect on zinc distribution. The nature of these interactions and which moieties may be involved cannot yet be discerned.
These experiments have been performed only at relatively low metal concentrations. From these results there appears to be differences between the effects of toxic metals (cadmium) compared to less essential metals (calcium, copper) upon zinc absorption. Whether a similar pattern exists upon raising luminal copper to toxic levels remains to be determined. The characterization of cadmium, copper, and calcium absorption would aid in clarifying interactions with zinc.
An investigation of amino acid effects upon zinc uptake also has been performed using the intestinal perfusion technique. These studies have described specific actions of histidine, cysteine, and surprisingly taurine upon zinc absorption. Despite chelation by histidine and cysteine, significant amounts of zinc were absorbed. Stereospecific actions of L-histidine were described and evidence was obtained suggesting the presence of transport of bis-histidine-zinc. Cysteine also had a stereospecific effect, acting to increase zinc levels in the blood. Of all the luminal constituents tested to date, only taurine acted on both intestinal and post-intestinal (blood and body) zinc accumulation rates. Taurine, which was initially included as a non-zinc binding control, and not believed to have an action on zinc uptake, stimulated intestinal but inhibited post-intestinal zinc accumulation. Again, further work is required to determine the mechanisms of these interactions.
Future Activities:
The last year of the grant will focus on characterization of Fugu ZnT-1, sequencing and bioinformatics processing of clones obtained from gene expression analysis of zinc exposed trout, studies of hormonal regulation of zinc uptake, and investigation of intracellular signaling pathways involved in modifying branchial zinc uptake.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:
water, freshwater, absorption, hardness, bioavailability, bioreactivity, metabolism, animal, fish, organism, cellular, enzymes, pollution, heavy metals, zinc, calcium, environmental biology, cell biology, integrative biology, environmental physiology, toxicology, zoology, uptake, regulation, endocrinology, acclimation, trace element, micronutrient, 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.