Grantee Research Project Results
Final 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 Amount: $462,981
RFA: Exploratory Research - Minority Institutions (1995) RFA Text | Recipients Lists
Research Category: Water , Land and Waste Management , Aquatic Ecosystems , Air
Objective:
This report includes a number of projects that were begun during the 3-year period covered by this EPA grant. Some of these projects were completed in this interval and others are ongoing at the present time and will serve as the beginning point of a new grant proposal to the EPA in the near future. The findings of each research project that was supported under this grant are summarized in this report and a list of publications and works in press that were generated during the 3-year grant cycle also is provided.
Summary/Accomplishments (Outputs/Outcomes):
Project 1. Calcium transport by hepatopancreatic brush border and basolateral plasma membranes. During the last three years, hepatopancreatic brush border membrane vesicle (BBMV) and basolateral membrane vesicle (BLMV) transport mechanisms for calcium ion 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 ocurred by the combination of three protein systems: (1) an electrogenic 1 Ca2+/1H+ antiporter that was amiloride-sensitive; (2) and electroneutral 1 Ca2+/2Na+ antiporter that was not affectd by amiloride; and (3) a membrane-potential-sensitive calcium channel that was inhibited by verapamil. The heavy metal, zinc, was shown to be a competitive inhibitor of 45Ca2+ uptake by BBMV, but the specific transport protein responsible for this interaction between the cations was not disclosed. Basolateral transport systems for 45Ca2+ included: (1) a low affinity Ca2+/Na+ exchanger of unknown stoichiometry; (2) a high affinity, vanadate sensitive, Ca2+-ATPase for pumping the cation out of the cell; and (3) a verapamil-sensitive calcium channel which may be important for Ca2+ uptake during premolt when large quantities of exoskeletal calcium enters the blood from the old shell and must be stored in hepatopancreatic granules or in stomach gastroliths.
Project 2. Calcium transport by hepatopancreatic mitochondria. Mitochondria from lobster hepatopancreas were purified by combining methods developed for isolation of these organelles from mammalian and crustacean tissues involving differential and Percoll-gradient centrifugation. Enrichment of the preparation was assessed using purification of enzyme markers and electron microscopic examination. 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+. 45Ca2+ efflux from mitochondria took place by a diltiazem-inhibited, electroneutral 2Na+/1Ca2+ antiporter and a diltiazem-insensitive 2H+/2Ca2+ antiporter. One or both antiporters were capable of exchanging preloaded 45Ca2+ for external Zn2+. Kinetic constants for uniporter mitochondrial calcium transport and the ruthnim 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.
Project 3. Confocal microscopy of calcium and heavy metal sequestration. A Bio-Rad MRC-600 confocal microscope was temporarily made available to this project to investigate calcium and heavy metal distribution and sequestration in isolated hepatopancreatic tubules and individual epithelial cells of the lobster. Calcium localization was determined using 60-min incubations in a physiological saline containing Pluronic F-127 in DMSO and Fluo-3, while BTC-5N was used to assess the distribution of heavy metals. The strongest indicator signals from isolated tubules were obtained from distal tip embryonic cells. In addition, a second strong signal occurred from cells in a wide, but defined, region proximal to the tip. Tubules exhibited a wide range of responses to the indicators, with some providing strong signals and others markedly less so. Tubules were dissociated into mixed cell suspensions of B-, R-, F-, and E-cells types using an isotonic citrate buffer. Autofluorescence was significantly less than signals from the indicators and appeared to be restricted to B-cell vacuoles. Punctate distribution of fluorescence from both dyes was shown in R- and F-cells suggesting possible sequestration of both calcium and metals in subcellular vacuoles or concretions. Cell viability was tested using propidium iodide and found to approximate 100 percent under experimental conditions employed. These preliminary observations suggest similar distributions and possible compartmentalization of calcium and heavy metals in hepatopancreatic tubule epithelial cells.
Project 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 brush border vesicles (BBMV) that were produced with a Mg2+ precipitation technique, and isotope uptake was monitored with a rapid filtration method. Osmotically reactive vesicles accumulated 65Zn more rapidly if they possessed an outwardly-directed H+ grdient and inside negative electrical potential difference (PD) than if they had equal pH values and were short-circuited. Decreasing inside pH or applying an inside electrically negative PD significantly increased 65Zn influx Jmax, but had minimal influence on the apparent affinity constant (e.g., Km) for metal uptake compared to control conditions. Addition of external Ca2+ (1 or 10 mM) did not influence the time course of 65Zn uptake, nor did this divalent cation enhance efflux by trans-stimulation of equilibrated 65Zn. 59Fe uptake was stimulated by an inside negative PD, inhibited by an inside positive PD, and reduced by 1 mM amiloride. 59Fe influx was unaffected by intravesicular Ca2+ (1 mM) suggesting a lack of trans-stimulation by this cation. Heavy metal accumulation by hepatopancreatic epithelial cells occurred by electrogenic, H+-dependent, transport that may be independent of Ca2+ uptake mechanisms.
Project 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 (Phen Green; Molecular Probes) was used to study epithelial Cu2+ uptake in isolated cellular vesicles and in E-, R-, F-, and B-cell types from the lobster hepatopancreas. Cells were separated by a centrifugal elutriation technique using a Beckman J5 high speed centrifuge adapted with an elutriation rotor and perfusion pump. All four hepatopancreatic cell types were separated using these instruments with a 90 to 95 percent purity and viability. In the first cycle F- and B-cells were isolated, and a mixture of two additional cell types and released cellular organelles was collected. This mixture was then used in a second cycle where cellular vesicles and E- and R-cells were next purified. These purified cell suspensions were then used in a series of experiments employing the fluorescent probe Phen Green to measure the kinetics of Cu2+ uptake and internalization within each cell type to ascertain differences between the cells in their heavy metal biology. Phen Green was loaded into cells by incubation at room temperature. The increase and stability of the fluroescence was monitored by spectrofluorometry in a Perkin Elmer recording spectrofluorometer. All four cell types accumulated copper from solution. 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+. This model of calcium-copper interaction is supported by the finding that addition of the calcium channel blocker, verapamil, to the external medium in the presence of external calcium, led to a sharp reduction in Cu2+ uptake by each cell type. Future experiments with this dye will extend these initial observations and clarify the nature between the two divalent cations.
Project 6. Ferrireductase activity in lobster hepatopancreatic brush border membrane vesicles and possible role in iron transport. Because of it possible role in heavy metal transport across epithelial membranes, a ferrireductase enzyme of hepatopancreatic brush border membrnae vesilces of the lobster was identified and characterized. Conversion of ferric chelates to ferrous iron was measured spectrophotometrically with the ferrous chromophore ferrozine. Conversion of ferricyanide to ferrocyanide was measured by the change in the difference in absorbance from 420 to 500 nm. Ferrireductase activity was dependent on NADH as a cofactor. NADPH could also serve as a cofactor in the ferrireductase reaction, but it was required at higher concentrations than NADH. The ferrireductase had the following reactivity toward different chelate substrates: ferricyanide (780 percent), ferric NTA (100 percent), ferric citrate (18.6 percent), ferric gluconate (16.3 percent), ferric EDTA (0 percent), and ferric HEDTA (0 percent). The activity of the ferrireductase was abolished by pretreatment of BBMV with heat (65 C; 5 min). Furthermore, there was no detectable activity when superoxide dismutase was incubated with the reaction mixture showing the requirement of the superoxide anion in the reaction. Ferrireductase activity was decreased to 32.8 percent when BBMV were pretreated with 500 m M p-chloromercuribenzoate. An established biological role of ferrireductase is the conversion of Fe3+ to Fe2+ in membranes of other organisms followed by the transmembrane transfer of the reduced iron form. The identification of this enzyme in lobster hepatopancreatic BBMV may indicate the presence of a transport protein accomodating 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.
Project 7. Effect of zinc on 3H-L-proline transport by lobster hepatopancreatic BBMV. Epithelial BBMV of the lobster hepatopancreas were prepared using a magnesium precipitation technique and employed in trnasport experiments designed to show the effects of external divalent cationic heavy metals on the uptake of 3H-L-proline. When BBMV were simultaneously exposed to Cd2+, Cu2+, Fe2+, Mn2+, or Zn2+ (2.5 mM) and 3H-L-proline (0.5 mM), amino acid uptake was significantly decreased by each metal. However, if a 30 min pre-incubation period with each metal was used before incubation of the vesicles with amino acid and metal, significant enhancement of 3H-L-proline transport took place. Zinc was the most stimulatory metal of those used. Proline influxes (1.0 and 2.5 mM) were hyperbolic functions of external [Zn] with a lower apparent zinc half saturation constant at the higher amino acid concentration. 3H-L-proline influx was a hyperbolic function of external [L-proline]. Exposure to 10 mM zinc significantly increased proline influx Jmax but had no effect on apparent Km (proline binding to the membranes). 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.
Project 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. To understand how heavy metals are sequestered into intracellular compartments we must first understand how the individual components of the insoluble precipitates gain access to the cytoplasm across both brush border and basolateral membranes of detoxifying epithelial cells. We have previously published papers describing plasma membrane transport systems for sulfate on the brush border membrane (SO4/Cl antiporter), and on the basolateral membrane (SO4/oxalate antiporter). In the present study a third SO4 transport system on the basolateral membrane is described. Transport of 35SO4 into BLMV was stimulated by an outwardly-directed bicarbonate gradient compared with gluconate-loaded vesicles. An inside-positive membrane potential stimulated sulfate/bicarbonate exchange, whereas an inside-negative membrane potential was inhibitory. Sulfate/sulfate exchange was not affected by alterations of transmembrane potential difference. External protons stimulated sulfate/bicarbonate exchange while proton gradients had no effect on this antiport process in BLMV. The stilbenes (SITS and DIDS) strongly inhibited SO4/HCO3 exchange. These results suggest that SO4/HCO3 exchange in hepatopancreatic BLMV occurred by an electrogenic carrier mechanism with a 1:1 flux ratio. This basolateral carrier system could provide a means by which sulfate is transported from the blood into hepatopancreatic epithelial cells during times of increased heavy metal load in these cells. With additional sulfate present intracellularly the detoxification mechanisms occurring in mitochondria and lysosomes which employ sulfate would have a greater supply of substrate and be better able to sequester the metals in these compartments.
Project 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. The centrifugal elutriation system described earlier was used to produce purified hepatopancreatic E-cells displaying less than 90 percent viability. Because the plasma membrane Na/H exchanger had previously been physiologically characterized in these cells, its mode of cellular regulation was first studied so that later investigations of transport systems accomodating heavy metals could be more accurately assessed. Intracellular pH (pHi) was monitored by loading cells with the fluorescent dye BCECF and placing the suspensions in a spectrofluorometer. Recovery from induced acid-loading was mediated by a Na-dependent, dimethylamiloride-sensitive proton efflux. Antiport activation was a sigmoidal function of pHi at values <7.0. Addition of 20 nM phorbol 12-myristate 13 acetate to cells suspended in a lobster physiological saline (pHo = 7.4) increased pHi from 7.2 to 7.5 over a 10 min interval. Phorbol ester induced activation of the Na/H antiporter was due to an increased affinity for internal H (apparent pK was shifted towards more alkaline values) at the level of an internal H-binding allosteric modifier site. Phorbol ester activation of the lobster NHE-like Na/H antiporter is consistent with regulatory pathways described for the vertebrate NHE-1. This study will lead the way to indentifying the regulatory processes responsible for uptake of heavy metals from luminal contents in hepatopancreatic epithelial cells.
Project 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 of this grant cycle. 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. Molecular biology can provide a means whereby the amino acid sequences of these proteins can be ascertained and the metal binding sites identified. 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. Recently, the gene sequence for the crustacean Na/H antiporter in crab gill epithelia. The methods used by these researchers were applied to the lobster hepatopancreatic Na/H exchanger to develop the techniques in my lab so that they could be generally applied in future years to the determination of heavy metal transporter genes. Degenate primers designed to anneal to conserved regions of the NHE gene family were employed in PCR experiments. Isolated mRNA from lobster hepatopancreas was reverse transcribed and the resulting cDNA used as template. One reaction using primers predicted to amplify a 700 bp fragment yielded a single band of the appropriate size upon agarose gel analysis. This cDNA fragment was sequenced using a fluorescent automated nucleic cid sequencer and homology to known cDNa sequences was assessed using the BLAST algorithm at the NIH GenBank. A sequence identity of 95 percent to the crab NHE homolog as well as significant homology to vertebrate NHE sequences suggested our 700 bp cDNA was a fragment of a lobster NHE homolog. This putative lobster NHE fragment was readiolabelled with 32P-ATP and used as a probe to screeen a lobster hepatopancreatic cDNA library. No positive clones to date have been found from the library screen which may be due to the very low abundance of NHE mRNA in hepatopancreas. Strategies to clone rare genes is currently 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.
Journal Articles on this Report : 14 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) |
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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) |
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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) |
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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) |
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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. |
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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. |
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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. |
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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. |
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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. |
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Klein MJ, Ahearn GA. Calcium transport mechanisms of crustacean hepatopancreatic mitochondria. Journal of Experimental Zoology 1999;283(2):147-159. |
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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. |
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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. |
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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. |
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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) |
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Supplemental Keywords:
toxicity, detoxification, heavy metals, crustaceans, lobster., 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.