Gregory A. Ahearn

Mechanisms of heavy-metal sequestration and detoxification in crustaceans: a review.

This review is an update of information recently obtained about the physiological, cellular, and molecular mechanisms used by crustacean organ systems to regulate and detoxify environmental heavy metals. It uses the American lobster, Homarus americanus, and other decapod crustaceans as model organisms whose cellular detoxification processes may be widespread among both invertebrates and vertebrates alike. The focus of this review is the decapod hepatopancreas and its complement of metallothioneins, membrane metal transport proteins, and vacuolar sequestration mechanisms, although comparative remarks about potential detoxifying roles of gills, integument, and kidneys are included. Information is presented about the individual roles of hepatopancreatic mitochondria, lysosomes, and endoplasmic reticula in metal sequestration and detoxification. Current working models for the involvement of mitochondrial and endoplasmic reticulum calcium-transport proteins in metal removal from the cytoplasm and the inhibitory interactions between the metals and calcium are included. In addition, copper transport proteins and V-ATPases associated with lysosomal membranes are suggested as possible sequestration processes in these organelles. Together with several possible cytoplasmic divalent and trivalent anions such as sulfate, oxalate, or phosphate, accumulations of metals in lysosomes and their complexation into detoxifying precipitation granules may be regulated by variations in lysosomal pH brought about by bafilomycin-sensitive proton ATPases. Efflux processes for metal transport from hepatopancreatic epithelial cells to the hemolymph are described, as are the possible roles of hemocytes as metal sinks. While some of the cellular processes for isolating heavy metals from general circulation occur in the hepatopancreas and are beginning to be understood, very little is currently known about the roles of the gills, integument, and kidneys in metal regulation. Therefore, much remains to be clarified about the organs and mechanisms involved in metal homeostasis in decapod crustaceans.

Ion Transport Processes of Crustacean Epithelial Cells

Epithelial cells of the gut, antennal glands, integument, and gills of crustaceans regulate the movements of ions into and across these structures and thereby influence the concentrations of ions in the hemolymph. Specific transport proteins serving cations and anions are found on apical and basolateral cell membranes of epithelia in these tissues. In recent years, a considerable research effort has been directed at elucidating their physiological and molecular properties and relating these characteristics to the overall biology of the organisms. Efforts to describe ion transport in crustaceans have focused on the membrane transfer properties of Na+/H+ exchange, calcium uptake as it relates to the molt cycle, heavy metal sequestration and detoxification, and anion movements into and across epithelial cells. In addition to defining the properties and mechanisms of cation movements across specific cell borders, work over the past 5 yr has also centered on defining the molecular nature of certain transport proteins such as the Na+/H+ exchanger in gill and gut tissues. Monovalent anion transport proteins of the gills and gut have received attention as they relate to osmotic and ionic balance in euryhaline species. Divalent anion secretion events of the gut have been defined relative to potential roles they may have in hyporegulation of the blood and in hepatopancreatic detoxification events involving complexation with cationic metals.

Cellular Mechanisms of Calcium Transport in Crustaceans

Exchanges of calcium and other ions of biological significance in crustaceans take place between the hemolymph and environment across epithelial cell layers of the integument, gills, gut, and antennal glands (kidneys). During most of the life cycle these exchanges are modest and are largely osmoregulatory, but during periodic molting large movements of ions, particularly calcium, occur between the organism and its environment; these movements must be closely regulated in order for the animal to grow and strengthen its new exoskeleton. This article describes, for the first time, the brush border and basolateral epithelial calcium transport processes located in the hepatopancreas of the American lobster, Homarus americanus, and discusses their potential roles in calcium balance during the molt cycle. Three independent brush border membrane transport proteins control the uptake of calcium from the gastrointestinal lumen: (1) an amiloride-sensitive, electrogenic cation antiporter exchanging 1 Ca²⁺ (or 2 Na⁺) for a single intracellular monovalent cation (H⁺ or Na⁺), (2) an amiloride-insensitive, probably electroneutral, cation antiporter exchanging 1 Ca²⁺ for 2 Na⁺ (or 2 H⁺), and (3) a verapamil- and amiloride-inhibited Ca²⁺ channel. Likewise, the epithelial basolateral membrane of this digestive organ possesses three calcium transfer processes for cation exchange between cytoplasm and blood: (1) a Ca²⁺/Na⁺ antiporter of unknown stoichiometry but of relatively low apparent Ca²⁺ binding affinity, (2) a high-afinity Ca²⁺-ATPase for cellular efflux, and (3) a verapamil-inhibited Ca²⁺ channel for possible calcium uptake from the blood during premolt. The kinetic properties of these six hepatopancreatic transporters and their potential general distribution to other ion regulatory epithelia in crustaceans are discussed in this article.

Cellular localization of calcium, heavy metals, and metallothionein in lobster (Homarus americanus) hepatopancreas

This investigation combines confocal microscopy with the cation-specific fluorescent dyes Fluo-3 and BTC-5N to localize calcium and heavy metals along the length of intact lobster (Homarus americanus) hepatopancreatic tubules and isolated cells. A metallothionein-specific antibody, developed in mollusks with cross-reactivity in crustaceans, showed the tissue-specific occurrence of this metal-binding protein in several organ systems in lobster and in single cell types isolated from lobster hepatopancreas. Individual lobster hepatopancreatic epithelial cell types were separated into pure single cell type suspensions for confocal and antibody experiments. Intact hepatopancreatic tubules showed high concentrations of both calcium and heavy metals at the distal tips of tubules where mitotic stem cells (E-cells) are localized. In addition, a concentrated distribution of calcium signal within isolated single premolt E-cells in solution was disclosed that might suggest an endoplasmic reticulum compartmentation of this cation within these stem cells. Both E- and R-cells showed significantly (P < 0.05) greater intracellular calcium concentrations in premolt than intermolt, suggesting the accumulation of this cation in these cells prior to the molt. Antibody studies with lobster tissues indicated that the hepatopancreas possessed 5-10 times the metallothionein concentration as other lobster organ systems and that isolated E-cells from the hepatopancreas displayed more than twice the binding protein concentrations of other cells of this organ or those of blood cells. These results suggest that crustacean hepatopancreatic stem cells (E-cells) and R-cells play significant roles in calcium and heavy metal homeostasis in this tissue. Interactions between the four hepatopancreatic cell types in this regulatory activity remain to be elucidated.

Copper transport by lobster (Homarus americanus) hepatopancreatic lysosomes.

Lysosomes are known centers for sequestration of calcium and a variety of heavy metals in many invertebrate tissues, and as a result of this compartmentalization these organelles perform important detoxification roles in the animals involved. The present investigation uses a centrifugation method to isolate and purify hepatopancreatic lysosomes from the American lobster, Homarus americanus. Purified lysosomal preparations were used to characterize membrane transport mechanisms in these organelles for transferring and sequestering cytoplasmic copper following its absorption across the plasma membrane from dietary constituents. The copper-specific fluorescent dye, Phen Green, was employed to quantify transmembrane fluxes of this metal as has been recently used to investigate copper movements across hepatopancreatic mitochondrial and plasma membranes. Results indicated the presence of a vanadate-sensitive, calcium-stimulated, copper ATPase in the membranes of these organelles that displayed high affinity carrier-mediated transport kinetics and may significantly contribute to organismic copper homeostasis. Together with a putative bafilomycin-sensitive V-ATPase in the membrane of the same organelles, importing hydrogen ions into the organellar interior, this copper ATPase may function as part of a physiological mechanism for precipitate formation between metallic cations and anions. These ionic precipitate complexes may then act as a sink for excess metals and thereby reduce the circulating concentrations of these elements.

Calcium translocations during the moulting cycle of the semiterrestrial isopod Ligia hawaiiensis (Oniscidea, Crustacea)

Terrestrial isopods moult first the posterior and then the anterior half of the body. During the moulting cycle they retain a significant fraction of cuticular calcium partly by storing it in sternal CaCO3 deposits. We analysed the calcium content in whole Ligia hawaiiensis and the calcium distribution between the posterior, the anterior ventral, and the anterior dorsal cuticle during four stages of the moulting cycle. The results indicate that: (1) overall, about 80% of the calcium is retained and 20% is lost with the exuviae, (2) in premoult 68% of the calcium in the posterior cuticle is resorbed (23% moved to the anterior ventral cuticle, 17% to the anterior dorsal cuticle, and the remaining 28% to internal tissues), (3) after the posterior moult 83% of the calcium in the anterior cuticle is shifted to the posterior cuticle and possibly to internal storage sites, (4) following the anterior moult up to 54% of the calcium in the posterior cuticle is resorbed and used to mineralise the new anterior cuticle. 45Ca-uptake experiments suggest that up to 80% of calcium lost with the anterior exuviae may be regained after its ingestion. Whole body calcium of Ligia hawaiiensis is only 0.7 times that of the fully terrestrial isopods. These terrestrial species can retain only 48% of whole body calcium, suggesting that the amount of calcium that can be retained by shifting it between the anterior and posterior integument is limited. We propose that fully terrestrial Oniscidea rely to a larger degree on other calcium sources like internal stores and uptake from the ingested exuviae.

3H-L-histidine and 65Zn2+ are cotransported by a dipeptide transport system in intestine of lobster Homarus americanus

SUMMARY The tubular intestine of the American lobster Homarus americanus was isolated in vitro and perfused with a physiological saline whose composition was based on hemolymph ion concentrations and contained variable concentrations of 3H-l-histidine, 3H-glycyl-sarcosine and 65Zn2+. Mucosa to serosa (M→S) flux of each radiolabelled substrate was measured by the rate of isotope appearance in the physiological saline bathing the tissue on the serosal surface. Addition of 1–50 μmol l–1 zinc to the luminal solution containing 1–50 μmol l–1 3H-l-histidine significantly (P<0.01) increased M→S flux of amino acid compared to controls lacking the metal. The kinetics of M→S 3H-l-histidine flux in the absence of zinc followed Michaelis–Menten kinetics (Km=6.2±0.8 μmol l–1; Jmax =0.09±0.004 pmol cm–2 min–1). Addition of 20 μmol l–1 zinc to the luminal perfusate increased both kinetic constants (Km=19±3 μmol l–1; Jmax=0.28±0.02 pmol cm–2 min–1). Addition of both 20 μmol l–1 zinc and 100 μmol l–1 l-leucine abolished the stimulatory effect of the metal alone (Km=4.5±1.7μ mol l–1; Jmax=0.08±0.008 pmol cm–2 min–1). In the absence of l-histidine, M→S flux of 65Zn2+ also followed the Michaelis–Menten relationship and addition of l-histidine to the perfusate significantly (P<0.01) increased both kinetic constants. Addition of either 50 μmol l–1 Cu+ or Cu2+ and 20 μmol l–1 l-histidine simultaneously abolished the stimulatory effect of l-histidine alone on transmural 65Zn2+ transport. Zinc-stimulation of M→S 3H-l-histidine flux was significantly (P<0.01) reduced by the addition of 100 μmol l–1 glycyl-sarcosine to the perfusate, as a result of the dipeptide significantly (P<0.01) reducing both l-histidine transport Km and Jmax. Transmural transport of 3H-glycyl-sarcosine was unaffected by the presence of either l-histidine or l-leucine when either amino acid was added to the perfusate alone, but at least a 50% reduction in peptide transport was observed when zinc and either of the amino acids were added simultaneously. These results show that 3H-l-histidine and 65Zn2+ are cotransported across the lobster intestine by a dipeptide carrier protein that binds both substrates in a bis-complex (Zn-[His]2) resembling the normal dipeptide substrate. In addition, the transmural transports of both substrates may also occur by uncharacterized carrier processes that are independent of one another and appear relatively specific to the solutes used in this study.

Heavy metal detoxification in crustacean epithelial lysosomes: role of anions in the compartmentalization process

SUMMARY Crustacean hepatopancreatic lysosomes are organelles of heavy metal sequestration and detoxification. Previous studies have shown that zinc uptake by lysosomal membrane vesicles (LMV) occurred by a vanadate- and thapsigargin-sensitive ATPase that was stimulated by a transmembrane proton gradient established by a co-localized V-ATPase associated with this organelle. In the present study, hepatopancreatic LMV from the American lobster Homarus americanus were prepared by standard centrifugation methods and 65Zn2+, 36Cl–, 35SO42– and 14C-oxalate2– were used to characterize the interactions between the metal and anions during vesicular detoxification events. Vesicles loaded with SO42– or PO43– led to a threefold greater steady-state accumulation of Zn2+ than similar vesicles loaded with mannitol, Cl– or oxalate2–. The stimulation of 65Zn2+ uptake by intravesicular sulfate was SO42– concentration dependent with a maximal enhancement at 500 μmol l–1. Zinc uptake in the presence of ATP was proton-gradient enhanced and electrogenic, exhibiting an apparent exchange stoichiometry of 1Zn+/3H+. 35SO42– and 14C-oxalate2– uptakes were both enhanced in vesicles loaded with intravesicular Cl– compared to vesicles containing mannitol, suggesting the presence of anion countertransport. 35SO42– influx was a sigmoidal function of external [SO42–] with 25 mmol l–1 internal [Cl–], or with several intravesicular pH values (e.g. 7.0, 8.0 and 9.0). In all instances Hill coefficients of approximately 2.0 were obtained, suggesting that 2 sulfate ions exchange with single Cl– or OH– ions. 36Cl– influx was a sigmoidal function of external [Cl–] with intravesicular pH of 7.0 and 9.0. A Hill coefficient of 2.0 was also obtained, suggesting the exchange of 2 Cl– for 1 OH–. 14C-oxalate influx was a hyperbolic function of external [oxalate2–] with 25 mmol l–1 internal [Cl–], suggesting a 1:1 exchange of oxalate2– for Cl–. As a group, these experiments suggest the presence of an anion exchange mechanism exchanging monovalent for polyvalent anions. Polyvalent inorganic anions (SO42– and PO43–) are known to associate with metals inside vesicles and a detoxification model is presented that suggests how these anions may contribute to concretion formation through precipitation with metals at appropriate vesicular pH.

Metabolic Effects of Sucralose on Environmental Bacteria

Sucralose was developed as a low cost artificial sweetener that is nonmetabolizable in humans. Sucralose can withstand changes in pH and temperature and is not degraded by the wastewater treatment process. Since the molecule can withstand heat, acidification, and microbial degradation, it is accumulating in the environment and has been found in wastewater, estuaries, rivers, and the Gulf Stream. Environmental isolates were cultured in the presence of sucralose looking for potential sucralose metabolism or growth acceleration responses. Sucralose was found to be nonnutritive and demonstrated bacteriostatic effects on all six isolates. This growth inhibition was directly proportional to the concentration of sucralose exposure, and the amount of the growth inhibition appeared to be species-specific. The bacteriostatic effect may be due to a decrease in sucrose uptake by bacteria exposed to sucralose. We have determined that sucralose inhibits invertase and sucrose permease. These enzymes cannot catalyze hydrolysis or be effective in transmembrane transport of the sugar substitute. Current environmental concentrations should not have much of an effect on environmental bacteria since the bacteriostatic effect seems to be consecration based; however, as sucralose accumulates in the environment, we must consider it a contaminant, especially for microenvironments.

Transepithelial d-glucose and d-fructose transport across the American lobster, Homarus americanus, intestine

SUMMARY Transepithelial transport of dietary d-glucose and d-fructose was examined in the lobster Homarus americanus intestine using d-[3H]glucose and d-[3H]fructose. Lobster intestines were mounted in a perfusion chamber to determine transepithelial mucosal to serosal (MS) and serosal to mucosal (SM) transport mechanisms of glucose and fructose. Both MS glucose and fructose transport, as functions of luminal sugar concentration, increased in a hyperbolic manner, suggesting the presence of mucosal transport proteins. Phloridizin inhibited the MS flux of glucose, but not that of fructose, suggesting the presence of a sodium-dependent (SGLT1)-like glucose co-transporter. Immunohistochemical analysis, using a goat anti-rabbit GLUT5 polyclonal antibody, revealed the localization of a brush border GLUT5-like fructose transport protein. MS fructose transport was decreased in the presence of mucosal phloretin in warm spring/summer animals, but the same effect was not observed in cold autumn/winter animals, suggesting a seasonal regulation of sugar transporters. Mucosal phloretin had no effect on MS glucose transport. Both SM glucose and SM fructose transport were decreased in the presence of increasing concentrations of serosal phloretin, providing evidence for the presence of a shared serosal GLUT2 transport protein for the two sugars. The transport of d-glucose and d-fructose across lobster intestine is similar to sugar uptake in mammalian intestine, suggesting evolutionarily conserved absorption processes for these solutes.