Final Report: Transport Mechanisms for Zinc Across Gill and Intestinal Epithelia of Fish

EPA Grant Number: R826104
Title: 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 Amount: $373,342
RFA: Exploratory Research - Environmental Biology (1997) RFA Text |  Recipients Lists
Research Category: Biology/Life Sciences , Ecosystems


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 general objective of the project was to delineate the pathways of zinc uptake in fish, and to characterize how zinc uptake is controlled. We hypothesized 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 the United States in terms of water quality criteria violations. Finally, because of their similarities in chemistry and mode of toxicity, zinc is a useful model substance for the more toxic metal, cadmium.

The four specific objectives were to: (1) characterize transfer of zinc across the basolateral membrane of the gill epithelium; (2) analyze mechanisms regulating apical entry of zinc and calcium in the gill epithelial cells; (3) investigate involvement of hormones in regulation of zinc homeostasis; and (4) physiologically characterize intestinal zinc uptake.

Summary/Accomplishments (Outputs/Outcomes):

Specific Objective 1: Characterization of zinc transfer across the basolateral membrane of the gill epithelium

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 proved to be markedly temperature sensitive, a hallmark of carrier-mediated transport.

In a complementary study, we cloned and sequenced a putative basolateral zinc exporter, ZnT-1, from a fish species. ZnT-1 is a zinc export protein first identified in mouse and rat. Low-stringency screening of a pufferfish (Fugu rubripes) cDNA library with a mouse ZnT-1 probe, resulted in the cloning and sequencing of the first fish ZnT-1 homologue. Sequence analysis exhibited 70 percent homology to rat and mouse ZnT-1. Extensive homology also extended to other zinc transporters identified in various organisms, showing that this transporter has been conserved in evolution from bacteria to vertebrates. Highest homology was observed in the proposed transmembrane domains. A predicted functional zinc recognition site, composed by a histidine-rich region in the large cytoplasmic loop between transmembrane domains four and five appears to be shorter in Fugu than in either mouse and rat. Two specific antibodies were raised against Fugu ZnT-1. Together with expression analysis, these will be used in future projects to characterize the physiology and expression of this zinc exporter.

Specific Objective 2: Analysis of mechanisms regulating apical entry of zinc and calcium in gill epithelial cells

Zinc acquisition in vertebrates is an active transcellular multistep process comprising zinc entry across the apical membrane, transport of zinc across the cell from apical to basolateral membrane, and finally, extrusion of zinc through the basolateral membrane. Zinc uptake is mediated by transporters, and entry of zinc into the gills is likely modulated by changes in properties and/or expression of such transporters at the apical surface. Zinc transporters of the ZIP family have been found responsible for zinc acquisition in yeast and plants. Two human ZIP transporters (hZIP1 and hZIP2) have been functionally characterized, but it is not known if these are responsible for intestinal zinc uptake. Semidegenerate polymerase chain reaction (PCR) was employed to identify putative zinc import proteins of the ZIP family from gills of zebra fish (Danio rerio) and rainbow trout. The 3' and 5' ends of the identified genes were amplified with RACE-PCR. Several putative zinc transporters were identified in gills of zebra fish (DrZIP1 and DrKE4) and rainbow trout (OmZIP1 and OmZIP2). Partial DrZIP1 (260aa) has 44 percent and 39 percent similarities to human ZIP1 and human ZIP2, respectively, and keeps the typical conserved topology and key functional amino acids postulated to be important to zinc transport. Partial OmZIP1 (127aa) and OmZIP2 (133aa) have 47 percent similarity to each other. OmZIP1 shares 96 percent similarity to DrZIP1. OmZIP2 shares 50 percent, 27 percent, and 45 percent similarities to hZIP1, hZIP2 and DrZIP1, respectively. Although DrKE4 (444aa) also keeps the conserved properties of ZIP family, it has potentially important structural differences. Continued research will establish whether or not these gene products indeed are responsible for zinc uptake in the teleost gill. Acute toxicity of zinc to fish is mediated by a hypocalcemia, generated by the competition between calcium and zinc at the apical membrane of the gill. Acclimation to waterborne zinc can occur, however, resulting in altered transport affinity and capacity of the entry route. Stanniocalcin is a putative endocrine mediator of this response, potentially acting through the cAMP/protein kinase A (PKA) pathway to modulate channel activity. Fingerling rainbow trout were exposed to 3.5 µM Zn(II) for a period of 120 hours. At regular time intervals, gill samples were taken and analysed for PKA, protein kinase C, and protein tyrosine kinase activity. Concurrently exposed and control fish gill calcium uptake parameters were monitored. Relative to the simultaneous control, zinc-exposed fish exhibited an enhanced PKA activity over the initial 24 hours. This correlated with decreased capacity and affinity for gill calcium uptake. No changes in protein tyrosine kinase or protein kinase C activity were detected. These results provide support for the hypothesis that the acclimation response of freshwater rainbow trout to waterborne zinc may be mediated by a stanniocalcin-dependent protein kinase A pathway.
Target cells in the gills as well as the entire organism may respond to metal exposure by reducing uptake, replacing target molecules, mobilizing detoxification mechanisms, and counteracting toxic effects of metals. Such biological variables can be studied in an integrative fashion by the use of functional genomics and proteomics; both functional genomics and proteomics have been used to cast light on the global gill response to waterborne zinc.

Rainbow trout fingerlings were exposed to 2.3 µM zinc for 2 weeks, using an untreated group as control. After 2 weeks, the fish were euthanized and the gills removed. Whole gill tissue mRNA was isolated from exposed and control trout. mRNA from fish of each treatment was used to prepare 32P-labelled cDNA, which was then differentially hybridized to a Fugu rubripes gill cDNA array (HGMP resource centre, Cambridge). Genes markedly upregulated in gills of rainbow trout during zinc exposure could broadly be grouped as being involved in: (1) paracellular integrity; (2) protein synthesis; (3) energy generation; and (4) immune response. In addition, a number of genes were identified with no previously described functions in vertebrates.

A proteomic approach using surface-enhanced laser desorption/ionization (SELDI) also was used to investigate the response of freshwater rainbow trout gill to waterborne zinc exposure. Rainbow trout fingerlings were exposed to 2.3 µM of zinc, and gills were harvested for proteomic analysis at 1, 3, and 6 days. Using three distinct "protein chip" types with differing protein binding properties, 42 proteins of molecular weight between 1,000 and 50,000 Daltons were identified that were absent in zinc-exposed fish, although 17 apparently unique proteins were found in zinc-treated fish. Many other proteins, although present in both control and experimental fish, were determined to alter in expression profile on waterborne zinc exposure. The use of a proteomic approach in environmental physiology and toxicology has enormous potential for examining an organisms response to a pollutant; however, analysis is somewhat limited due to the lack of protein databases for organisms used as models for toxicant response.

Specific Objective 3: Involvement of hormones in regulation of Zn homeostasis

We studied the possible regulation of zinc by cortisol, prolactin, and the vitamin D3 metabolite, 1,25-(OH)2D3 . Both 1,25-(OH)2 D3 and prolactin increased branchial zinc uptake substantially relative to sham-treated controls. In either case, the enhanced zinc uptake was apparent 168 hours after hormone injection, with no obvious effect at 24 or 72 hours. The stimulatory effect of 1,25-(OH)3 D3 supports our previous finding that zinc partially shares the uptake pathway with Ca across the gill. The response caused by prolactin is consistent with data from mammalian cells suggesting that expression of ZIP may be regulated by prolactin. In light of these results, a partial cDNA sequence of a 1,25-(OH)3 D3 receptor (OmVDR, 176aa) belonging to the nuclear receptor superfamily was cloned from rainbow trout gill. The cDNA sequence shares 82 percent, 84 percent, and 82 percent similarities to zebra fish VDR, flounder VDRa, and flounder VDRb, respectively. Thus, the presence of VDR in gill of rainbow trout indicates that the observed effect of 1,25-(OH)3 D3 on branchial Zn influx is specific.

Specific Objective 4: Physiological characterization of intestinal Zn uptake

For aquatic organisms, zinc is both an essential nutrient and an environmental contaminant. The intestine is potentially the most important route of zinc absorption, yet little is known regarding this uptake pathway for zinc in fish. Furthermore, the diet is a complex mixture that has the potential to influence the uptake and metabolism of luminal zinc, and consequently its nutrition and toxicity.

The physiological mechanisms of intestinal zinc uptake in freshwater rainbow trout were investigated using an in vivo perfusion technique. Only a saturable component of zinc uptake, with a K0.5 of 309 µM, and a Jmax of 933 nmol kg-1hr-1, was described, characterizing the intestine as a low affinity, high capacity zinc absorption pathway. Physiological mechanisms appeared to regulate zinc uptake. Intestinal mucus was one important regulatory locus, promoting zinc uptake at low concentrations yet buffering the animal against high luminal zinc loads. Regulatory mechanisms also seemed to limit subepithelial zinc accumulation. There was a higher proportion of loosely associated zinc at higher perfused concentrations, attributed to saturation of the uptake process or efflux from the subepithelium. Two distinct pathways for passage of zinc across the epithelium were discerned, with postintestinal transfer possibly mediated by sulphydryl groups as illustrated by N-ethylmaleimide inhibition of zinc transfer to extra-intestinal compartments.

The effects of amino acids and hydrominerals on intestinal zinc uptake also were studied. The presence of histidine, cysteine, and taurine had distinct modifying actions on quantitative and qualitative zinc absorption, compared to perfusion of zinc alone. Alterations in zinc transport were not correlated to changes in free zinc ion. The chemical nature of the zinc-amino acid chelate, rather than the chelation itself, appeared to have the most important influence on zinc absorption. L-histidine, despite a strong zinc-chelating effect, maintained quantitative zinc uptake at control (zinc alone) levels. This effect correlated with the formation of Zn(His)2 species. D-histidine at a luminal concentration of 100 mM significantly enhanced subepithelial zinc accumulation, but reduced the fraction of zinc that was retained and absorbed by the fish. Collectively, the results indicate that there is a Zn(His)2 - mediated pathway for intestinal uptake in rainbow trout. L-cysteine specifically stimulated the accumulation of zinc postintestinally, an effect attributed to enhanced zinc accumulation in the blood. Taurine increased subepithelial zinc accumulation, but decreased the passage of zinc to postintestinal compartments. Amino acids are proposed to have important roles in modifying intestinal zinc uptake with potential implications for environmental toxicity.

Luminal cadmium and copper had specific, yet distinct, antagonistic effects on intestinal zinc absorption in rainbow trout. Copper significantly reduced the proportion of zinc absorbed from 11 percent to 6 percent of the total zinc perfused, concomitantly limiting the passage of zinc into the circulation and beyond. Conversely, cadmium decreased subepithelial zinc accumulation, with rates falling to 29 nmol g-1 hour-1 from control (zinc alone) values of 53 nmol g-1 hour-1. Calcium had a similar action to copper, also reducing postintestinal zinc accumulation from 0.06 to 0.02 nmol g-1 hour-1, an effect attributed to interactions between calcium and the zinc uptake pathway. In addition to these effects, luminal composition also had a marked influence on epithelial response to zinc; calcium, copper, and magnesium all greatly reduced zinc-induced mucus secretion, whereas cadmium significantly increased mucus secretion. It is proposed that these modifications were related to the essentiality of each element and their potential mechanisms of uptake. Despite changes at the epithelium, the postepithelial absorption of zinc was dependent mainly on the nature of the competing cation. Intestinal saline ion substitution experiments suggested a potential link of potassium ion efflux to zinc uptake. It was apparent that luminal composition had a significant influence on zinc absorption, highlighting the importance of studying the mechanism of metal uptake in complex mixtures to generate physiologically and environmentally relevant data.

The possibility of different modes of Zn uptake in freshwater and marine fish was considered. Intestinal zinc uptake was studied in two marine teleosts, the gulf toadfish (Opsanus beta) and a zinc hyper-accumulating species, the squirrelfish (Holocentrus adscensionis). Two in vitro techniques (in vitro perfusion and intestinal sacs) and one in vivo technique (in situ bolus) were employed. Female squirrelfish exhibited significantly enhanced epithelial zinc uptake and hepatic zinc accumulation, confirming this animals' extraordinary sex-specific zinc metabolism. Zinc uptake in toadfish was biphasic with respect to zinc concentration. This pattern was independent of the technique used. Postintestinal zinc uptake, however, was highly dependent on technique. The decreased relative uptake with in vivo methodology and the altered proportion of post-intestinal zinc accumulation with concentration suggested regulation of postintestinal zinc transfer. The results were used to reinterpret previous findings of zinc uptake in freshwater fish and allowed a critique of techniques used to study intestinal metal uptake.

Journal Articles on this Report : 7 Displayed | Download in RIS Format

Other project views: All 27 publications 7 publications in selected types All 7 journal articles
Type Citation Project Document Sources
Journal Article 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
Journal Article 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
Journal Article Farag AM, Woodward DE, Goldstein JN, MacConnell E, Hogstrand C, Barrows FT. Dietary effects of metals-contaminated invertebrates from the Coer d'Alene River, Idaho, on cutthroat trout. Transactions of the American Fisheries Society 1999;128(4):578-592. R826104 (Final)
not available
Journal Article Glover CN, Hogstrand C. Amino acid modulation of in vivo intestinal zinc absorption in freshwater rainbow trout. Journal of Experimental Biology 2002;205(1):151-158. R826104 (Final)
not available
Journal Article Glover CN, Hogstrand C. In vivo characterisation of intestinal zinc uptake in freshwater rainbow trout. Journal of Experimental Biology 2002;205(1):141-150 R826104 (Final)
not available
Journal Article Hogstrand C, Verbost PM, Bonga SEW. Inhibition of human erythrocyte Ca2+-ATPase by Zn2+. Toxicology 1999;133(2-3):139-145. R826104 (Final)
not available
Journal Article Hollis L, Hogstrand C, Wood CM. Tissue-specific cadmium accumulation, metallothionein induction, and tissue zinc and copper levels during chronic sublethal cadmium exposure in juvenile rainbow trout. Archives of Environmental Contamination and Toxicology 2001; 41(4):468-474. 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, functional genomics, proteomics, intracellular signalling., RFA, Scientific Discipline, Toxics, Waste, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, National Recommended Water Quality, exploratory research environmental biology, Ecosystem/Assessment/Indicators, Chemical Mixtures - Environmental Exposure & Risk, Ecosystem Protection, Zoology, Ecological Effects - Environmental Exposure & Risk, Environmental Microbiology, Ecological Effects - Human Health, Biochemistry, Ecology and Ecosystems, Ecological Indicators, cell transformation, endocrine disrupting chemical, transport contaminants, endocrine disruptors, toxic metals, cellular mechanisms, Zinc, aquatic ecosystems, water quality, amino acids, cadmium, fish , acclimation processes, rainbow trout

Relevant Websites: Exit

Progress and Final Reports:

Original Abstract
  • 1998 Progress Report
  • 1999