Grantee Research Project Results
1998 Progress Report: Cellular Mechanism of Heavy Metal Detoxification in Crustaceans
EPA Grant Number: R823068Title: Cellular Mechanism of Heavy Metal Detoxification in Crustaceans
Investigators: Ahearn, Gregory A.
Institution: University of Hawaii at Honolulu
EPA Project Officer: Packard, Benjamin H
Project Period: August 1, 1995 through August 1, 1998
Project Period Covered by this Report: August 1, 1997 through August 1, 1998
Project Amount: $462,981
RFA: Exploratory Research - Minority Institutions (1995) RFA Text | Recipients Lists
Research Category: Water , Land and Waste Management , Aquatic Ecosystems , Air
Objective:
The objective of this research project is to investigate the cellular mechanisms by which crustaceans sequester and detoxify environmental metals.
Progress Summary:
Progress has been made on the following 10 studies:
1. Calcium Transport by Hepatopancreatic Brush Border and Basolateral Plasma Membranes: During the last 3 years, hepatopancreatic brush border membrane vesicle (BBMV) and basolateral membrane vesicle (BLMV) transport mechanisms for calcium ions were studied as part of a larger project to examine the mechanisms for regulating divalent cations by the gastrointestinal tract in crustaceans. Calcium uptake across the luminal hepatopancreatic membrane into the epithelial cells of this organ occurred by the combination of three protein systems: (1) an electrogenic 1Ca2+/1H+ antiporter that was amiloride-sensitive; (2) an electroneutral 1Ca2+/2Na+ antiporter that was not affected by amiloride; and (3) a membrane-potential-sensitive calcium channel that was inhibited by verapamil.
2. Calcium Transport by Hepatopancreatic Mitochondria: Mitochondria from lobster hepatopancreas were purified. 45Ca2+ uptake into these organelles occurred by an electrogenic, ruthenium red-inhibited uniporter process that was sensitive to cytoplasmic pH and heavy metals, such as Zn2+. Kinetic constants for uniporter mitochondrial calcium transport and the ruthenium red inhibitory constant of this transporter were presented. Models of mitochondrial uptake and release were developed, as these activities relate to transient sequestration and transcellular calcium transfers during the molt cycle.
3. Confocal Microscopy of Calcium and Heavy Metal Sequestration: A confocal microscope was used to investigate calcium and heavy metal distribution and sequestration in isolated hepatopancreatic tubules and individual epithelial cells of the lobster. Preliminary observations suggest similar distributions and possible compartmentalization of calcium and heavy metals in hepatopancreatic tubule epithelial cells.
4. 65Zn and 59Fe Uptake by Lobster Hepatopancreatic Epithelial Cells Occur by Electrogenic, Proton-Dependent Transport Processes: Transport mechanisms for 65Zn and 59Fe of hepatopancreatic epithelial cells of American lobster (Homarus americanus) were investigated using BBMVs that were produced with a Mg2+ precipitation technique. Isotope uptake was monitored with a rapid filtration method. Heavy metal accumulation by hepatopancreatic epithelial cells occurred by electrogenic, H+-dependent transport that may be independent of Ca2+ uptake mechanisms.
5. Development of an Efficient Centrifugal Elutriation Method to Isolate and Purify Distinct Cell Populations From the Crustacean Hepatopancreas and Use in Copper Transport Studies: A fluorescent probe was used to study epithelial Cu2+ uptake in isolated cellular vesicles and in E-, R-, F-, and B-cell types from the lobster hepatopancreas. The uptake process only occurred when external calcium was present in the incubation medium. In the presence of external Ca2+, Cu2+ uptake was a hyperbolic function of external copper concentration. The stimulation of copper uptake by external calcium suggests that calcium enters the cells, perhaps by a channel, and then acts as an antiport substrate to exchange with external Cu2+. Future experiments will extend these initial observations and clarify the nature between the two divalent cations.
6. Ferrireductase Activity in Lobster Hepatopancreatic BBMVs and Possible Role in Iron Transport: Because of its possible role in heavy metal transport across epithelial membranes, a ferrireductase enzyme of the lobster hepatopancreatic BBMVs was identified and characterized. The identification of this enzyme in lobster hepatopancreatic BBMVs may indicate the presence of a transport protein accommodating the uptake of reduced iron from dietary contents in this animal. Future experiments will ascertain the presence and nature of such an iron transport protein and its potential role in the heavy metal biology of this animal.
7. Effect of Zinc on 3H-L-Proline Transport by Lobster Hepatopancreatic BBMV: Epithelial BBMVs of the lobster hepatopancreas were prepared using a magnesium precipitation technique and employed in transport experiments designed to show the effects of external divalent cationic heavy metals on the uptake of 3H-L-proline. Zinc was the most stimulatory metal of those used. In the presence of 0.5 mM L-pipecolate, zinc-stimulated, carrier-mediated, 3H-L-proline influx was abolished. Alternative models accounting for zinc-enhanced 3H-L-proline influx by the L-pipecolate-sensitive IMINO transport system in these membranes will be evaluated in future experiments.
8. Sulfate/Bicarbonate Antiport by Lobster Hepatopancreatic Basolateral Membrane Vesicles: During heavy metal detoxification in invertebrate epithelial cells, cationic metal ions such as zinc, copper, and iron are integrated with divalent anions such as sulfate to form an insoluble precipitate that is stored in lysosomes or mitochondria. One goal of this research program is to clarify the nature of this sequestration phenomenon.
9. Physiological Regulatory Mechanisms for a Hepatopancreatic Ion Transport Process: During this research program, a project was begun to examine how cells regulate the transmembrane transport of ions by way of carrier-mediated transporters. This study will lead the way to identifying the regulatory processes responsible for uptake of heavy metals from luminal contents in hepatopancreatic epithelial cells.
10. Molecular Biology of Ion Transporting Proteins of the Crustacean Hepatopancreas: There have been three hepatopancreatic epithelial divalent cation transporting proteins identified and physiologically characterized over the last 3 years. Two of these proteins occur on the epithelial brush border (Ca/H or Ca/Na antiporters), and the third is localized on the epithelial basolateral membrane (Ca-ATPase). The heavy metals zinc and iron interact with all three of these proteins either in an inhibitory fashion or are transported across the respective membrane in place of calcium or sodium. Very little is known about the nature of the binding sites on these proteins for their normal substrates or for the heavy metals. Common characteristics of these binding sites might be present and a knowledge of these characteristics might be used to reduce metal toxicity in aquatic animals that come into contact with these agents. The gene nucleotide sequences coding for these proteins in vertebrates are known and can be accessed from the literature. Based on these known sequences, the molecular biology technique of Polymerase Chain Reaction (PCR) can be used to generate the unknown sequences of the genes coding for the metal transporting proteins in crustacean hepatopancreas. Once the gene nucleotide sequences are known, one can deduce the amino acid sequences of the protein product. Strategies to clone rare genes currently are being applied to this system to obtain a complete NHE cDNA clone from the lobster hepatopancreas. These methods will be later applied to cloning a gene responsible for producing a heavy metal transport protein in hepatopancreatic tissue.
Future Activities:
Future activities will include completing the studies described in this report and conducting any follow-on experiments.
Journal Articles on this Report : 15 Displayed | Download in RIS Format
Other project views: | All 33 publications | 15 publications in selected types | All 15 journal articles |
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Ahearn GA. The invertebrate electrogenic 2Na+/1H+ exchanger: polyfunctional epithelial workstation. Physiology 1996;11(1):31-35. |
R823068 (1998) R823068 (Final) |
Exit |
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Ahearn GA, Zhuang Z. Cellular mechanisms of calcium transport in crustaceans. Physiological Zoology 1996;69(2):383-402. |
R823068 (1998) R823068 (Final) |
Exit |
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Ahearn GA, Duerr JM, Zhuang Z, Brown RJ, Aslamkhan A, Killebrew DA. Ion transport processes of crustacean epithelial cells. Physiological and Biochemical Zoology 1999;72(1):1-18. |
R823068 (1998) R823068 (Final) |
Exit |
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Aslamkhan AG, Ahearn GA. Iron uptake by hepatopancreas brush border membrane vesicles (BBMV) of the lobster (Homarus americanus). Journal of Experimental Zoology Part A: Comparative Experimental Biology 2003;295A(2):145-150. |
R823068 (1998) |
Exit |
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Chavez-Crooker P, Garrido N, Ahearn GA. Copper transport by lobster hepatopancreatic epithelial cells separated by centrifugal elutriation:measurements with the fluorescent dye Phen Green. Journal of Experimental Biology 2001;204(8):1433-1444. |
R823068 (1998) R823068 (Final) |
Exit Exit |
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Duerr JM, Ahearn GA. Phorbol ester activation of an NHE-like electroneutral Na+/H+ antiporter in isolated E-cells of lobster (Homarus americanus) hepatopancreas. Journal of Experimental Zoology 1998;281(2):97-108. |
R823068 (1998) R823068 (Final) |
Exit |
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Duerr J, Ahearn G. Characterization of a basolateral electroneutral Na+/H+ antiporter in Atlantic lobster (Homarus americanus) hepatopancreatic epithelial vesicles. Journal of Experimental Biology 1996;199(3):643-651. |
R823068 (1998) R823068 (Final) |
Exit Exit |
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Gerencser GA, Cattey MA, Ahearn GA. Sulfate/oxalate exchange by lobster hepatopancreatic basolateral membrane vesicles. American Journal of Physiology-Regulatory Integrative and Comparative Physiology 1995;269(3):R572-R577. |
R823068 (1998) R823068 (Final) |
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Gerencser GA, Ahearn GA, Cattey MA. Antiport-driven sulfate secretion in an invertebrate epithelium. Journal of Experimental Zoology 1996;275(4):269-276. |
R823068 (1998) R823068 (Final) |
Exit |
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Gerencser GA, Ahearn GA, Cattey MA. Sulfate/bicarbonate antiport by lobster hepatopancreatic basolateral membrane vesicles. Journal of Experimental Zoology 1999;284(2):158-167. |
R823068 (1998) R823068 (Final) |
Exit |
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Klein MJ, Ahearn GA. Calcium transport mechanisms of crustacean hepatopancreatic mitochondria. Journal of Experimental Zoology 1999;283(2):147-159. |
R823068 (1998) R823068 (Final) |
Exit |
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Monteilh-Zoller MK, Zonno V, Storelli C, Ahearn GA. Effects of zinc on L-[3H]proline uptake by lobster (Homarus americanus) hepatopancreatic brush-border membrane vesicles. Journal of Experimental Biology 1999;202(21):3003-3010. |
R823068 (1998) R823068 (Final) |
Exit Exit |
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Zhuang Z, Ahearn GA. Energized Ca2+ transport by hepatopancreatic basolateral plasma membranes of Homarus americanus. Journal of Experimental Biology 1998;201(2):211-220. |
R823068 (1998) R823068 (Final) |
Exit Exit |
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Zhuang Z, Duerr JM, Ahearn GA. Ca2+ and Zn2+ are transported by the electrogenic 2Na+/1H+ antiporter in echinoderm gastrointestinal epithelium. Journal of Experimental Biology 1995;198(5):1207-1217. |
R823068 (1998) R823068 (Final) |
Exit Exit |
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Zhuang Z, Ahearn GA. Ca2+ transport processes of lobster hepatopancreatic brush-border membrane vesicles. Journal of Experimental Biology 1996;199(5):1195-1208. |
R823068 (1998) R823068 (Final) |
Exit Exit |
Supplemental Keywords:
heavy metals, detoxification, calcium transport, heavy metal sequestration, heavy metal detoxification, crustaceans., RFA, Scientific Discipline, Toxics, Ecosystem Protection/Environmental Exposure & Risk, National Recommended Water Quality, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, Environmental Chemistry, Chemistry, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, Electron Microscopy, Ecological Indicators, cell transformation, electron microscope, marine ecosystem, detoxification, isotopic flux, chemical contaminants, cellular mechanisms, Zinc, toxicity, microprobe, "Minority Institutions", cadmiumProgress 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.