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Iron acquisition by teleost fish
Comparative Biochemistry and Physiology Part C 135 (2003) 97–105
Review
Iron acquisition by teleost fish Nicolas Burya,*, Martin Grosellb a
King’s College London, School of Health and Life Sciences, Franklin Wilkins Building, 150 Stamford Street, London SE1 9NN, UK b Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 33149-1098 Miami, FL, USA Received 31 October 2002; received in revised form 21 January 2003; accepted 21 January 2003
Abstract Iron is a vital micronutrient for teleost fish, being an integral component of proteins involved in cellular respiration and oxygen transfer. However, in excess iron is toxic, and fish need to balance uptake to prevent deficiency vs. potential toxicity. This review assesses the current physiological and molecular knowledge of the mechanisms of iron acquisition in the teleost fish. It focuses on freshwater teleost fish when assessing the gill as a possible site for iron acquisition, and includes a summary of geochemical processes that govern aquatic iron bioavailability. It focuses on marine teleost fish for assessing the mechanism of intestinal iron uptake. Physiological evidence indicates that iron preferentially crosses the apical membrane of both the gills and intestine in the ferrous (Fe2q) state. Molecular evidence supports this, demonstrating the presence of homologues in fish to the large Slc 11a family of evolutionary conserved proteins linked to Fe2q transport. This symporter is probably linked to a reductase, which reduces either ferric (Fe3q ) or organic complexed iron to Fe2q prior to uptake. 2003 Elsevier Science Inc. All rights reserved. Keywords: Divalent metal transporter; Ferroportin; Ferric reductase; Siderophore; Zebrafish; European flounder; Desferrioxamine; Metal transport; Copper
1. Introduction Iron is one of the most abundant metals on the earth and is essential to almost all organisms. This is because iron’s flexible redox activity enables it The present (N. Bury and M. Grosell: Iron acquisition by teleost fish) and subsequent paper (P. Chavez-Crooker, N. Garrido, P. Pozo, and G.A. Ahearn: Copper transport by lobster (Homarus americanus) hepatopancreatic lysosomes) originated from presentation given in the Homeostasis of essential yet toxic metals symposium, organizers Nic Bury and Martin Grosell, sponsored by the Society for Experimental Biology and the International Copper Association (ICA) at the American Physiological Society Conference – The Power of Comparative Physiology: Evolution, Integration and Application, San Diege, USA, 24–28th August 2002. *Corresponding author. Tel.: q44-2078484091; fax: q442078484500. E-mail address: [email protected] (N. Bury).
to be an integral component of cellular respiration, and in addition its positioning in haemoglobin enhances the oxygen carrying capacity of the blood. However, in excess iron can be toxic. At the cellular level iron catalyses the Fention reaction (Eq. (1)) resulting in the generation of free radical species, including hydroxyl radicals, that can potentially cause cellular oxidative damage. Fe2qqH2O2™Fe3qqOHyqOH•
(1)
Intracellular concentrations of iron are maintained stable via the regulation of iron import, export, as well as altering the cellular iron buffering capacity. The protein ferritin acts as a intracellular iron ‘soak’, taking up ferrous iron (Fe2q), oxidising Fe2q to ferric iron (Fe3q) and incorporating Fe3q into its molecule. Within the cell
1532-0456/03/$ - see front matter 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1532-0456Ž03.00021-8
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intracellular iron levels are monitored by iron regulatory proteins (IRP). In instances of reduced cellular iron concentrations the apo-IRP binds to iron regulatory elements (IRE) in the untranslated regions of the ferritin gene preventing translation; at times of excess iron the IRP is inactivated, and levels of ferritin increase (Crichton et al., 2002). cDNA encoding for the Atlantic salmon (Salmo salar) ferritin protein possess consensus IREs in the upstream untranslated region (Andersen et al., 1995), suggesting its involvement in the regulation of cellular iron concentrations. At the whole organism level there is no known regulated excretory pathway for iron; however, in vertebrates iron is lost via the bile as a consequence of haemoglobin breakdown and via sloughing of the intestinal epithelium. Consequently, iron homeostasis is solely maintained via the regulation of absorption from the diet. In fish there are two potential sites for metal uptake, across the intestinal (dietary borne) or branchial epithelium (water borne). To prevent metal deficiency or toxic excess fish need to judge their metal needs, assess the available levels of metal in the water or diet and then integrate regulation of branchial andyor intestinal import accordingly to meet demand. This critical minireview will focus on teleost fish iron requirements and the molecular and physiological evidence for potential mechanisms of iron acquisition at the branchial and intestinal epithelium. 2. Teleost fish iron requirements Iron is routinely added, as iron salts, to fish feeds in aquaculture, but the precise daily iron turnover rates for teleost fish are presently unknown. The concentrations of iron in the diet required to prevent signs of iron deficiency (i.e. perturbed haematological parameters) for salmonids are 60–100 mg Fe kgy1 (Andersen et al., 1997), channel catfish (Ictalurus punctatus) 30 mg Fe kgy1 (Gaitlin and Wilson, 1986), and puffer fish (Takifugu rubripes) 90–140 mg Fe kgy1 (Zibdeh et al., 2001). In the salmonid fish farming industry the levels of iron previously incorporated into the diets was considerably higher, but a reduction was implemented following evidence that excess iron may be responsible for the disease winter ulcers (Salte et al., 1994). A recent investigation estimated the daily loss of iron by the freshwater zebrafish to be 14 mg kgy1 dayy1
(Bury and Grosell, unpublished data), which compares favourably with the estimated loss of iron by humans (28 mg kgy1 dayy1 based on a loss of 1–2 mg Fe per day by a 70 kg male, Conrad et al., 1999). If we assume that fish eat approximately 2% of their body weight of food a day (Jobling, 1995), then the diet of a 1 kg fish must contain between 1.2 and 2 mg of iron a day. The uptake efficiency of iron from this diet required to balance iron loss would have to be in the order of 0.7– 1.2%. This is considerably lower than an uptake efficiency of between 5.5 and 16.6% for humans, based on the daily ingest rate of between 12 and 18 mg Fe (Crichton et al., 2002). The disparity may reflect the form in which iron is present in the feed. Andersen et al. (1997) showed that if iron is added as a haem complex then it is more readily bioavailable to Atlantic salmon than ferrous salts. It will be of interest to see if the daily iron requirements for commercially important species, such as the salmonids are similar to the zebrafish. Modifying the diet to include more readily bioavailable forms of iron will improve the quality of fish feeds for the aquaculture industry. 3. Bioavailability of aquatic iron The chemical form of iron (Fe3q, Fe2q, Feinorganic or -organic complexes), which the transport epithelium encounters will determine the mechanism of the apical membrane iron uptake pathway. (Reference to the intestinal chemistry will be presented in the section covered by Intestinal iron uptake in marine teleost fish). Fe2q is more readily available than Fe3q, but in oxic aquatic environments and at circumneutral pH Fe2q is readily oxidised to Fe3q. In addition, the bioavailability of iron to unicellular organisms in freshwater is far greater than that of seawater, and in the open oceans iron is the limiting nutrient of primary productivity (Martin et al., 1991). In freshwater the oxidation rate of Fe2q is considerably slower (hours) than in seawater (minutes) (Gunnars et al., 2002), and in these oxic environments iron is predominantly found as colloidal hydrous iron oxides that are not biologically available (Stumm and Morgan, 1996; Lienemann et al., 1999). The physio-chemical characteristics of seawater (high cation concentrations, alkaline pH) result in colloid aggregation (Forsgren et al., 1996), resulting in the half-time to removal of iron colloidal particles in seawater being considerably
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faster (;75 h), compared to freshwater (;1000 h) (Gunnars et al., 2002). This will in turn cause a slower turnover of iron in freshwater ecosystems. Fe(III) oxyhydroxide colloids, which may also be complexed with organic matter (Tipping, 1981), settle out and the reducing environs of the anoxic zone or sediment results in Fe(III) reduction and Fe(II) leaches from the sediment generating an iron cycle (Stumm and Morgan, 1996). In the summer months stratification of eutrophic lakes may result in an oxic zone extending only a few metres below the surface, and during the autumn the breakdown of the thermocline as the lake cools results in the mixing of the bottom nutrient and iron rich waters. Despite the propensity for iron oxidation in the freshwater environment it is not a limiting factor in lotic or lentic phytoplankton growth (Hyenstrand et al., 1999). Based on these observations it would appear that there is a greater potential for iron uptake by the gills of freshwater fish compared to marine fish, but that the diet is probably the most important source of iron in both environments. An important aspect of iron speciation in aquatic ecosystems, that has often been overlooked, is the presence of inorganic sulphides, which, despite being present at relatively low concentrations in oxic waters (-1 mM), form complexes with iron that may account for 20% of the total dissolved iron content (Rozan et al., 2000). At present there is no evidence that iron sulphide is bioavailable to fish. But, interestingly, silver when complexed to sulphide (Bowles et al., 2002) in the aquatic environment enhances silver bioavailability to the water flea (Daphnia magna) (Bianchini et al., 2002). The generally low concentrations of free iron in freshwater and marine environment has resulted in a number of organisms, including bacteria, yeast (Winklemann, 2002), and phytoplankton (Wilhelm and Trick, 1994; Weger et al., 2002), evolving specialised mechanisms for mobilising and sequestering iron. These organisms produce and secrete secondary metabolites, termed siderophores, with exceptionally high binding affinity (;log K 19) for iron (Witter et al., 2000). Siderophore production is not just restricted to the aquatic environment; strategy II plants also secrete them to aid iron uptake from the soil (Guerniot, 2001). Siderophores maintain iron in solution and these organisms have specialised membrane transport processes for the uptake of the iron–siderop-
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hore complex (Wilhelm and Trick, 1994; Winklemann, 2002; Cowart, 2002). It has been estimated that a large proportion of iron in solution, particularly in the open ocean, will be found as organic-Fe complexes (Wu and Luther, 1995). These will include the siderophore–Fe complexes, as well as Fe that originates from intracellular porphyrins, which are released upon cell death (Rue and Bruland, 1995; Hutchins et al., 1999). This organic chelated iron is available to marine eukaryote phytoplankton (Hutchins et al., 1999) probably via a membrane bound ferric reductase (Jones et al., 1987). However, other possible uptake routes of iron that are associated with organic matter may exist for other eukaryotes. The bakers yeast Saccharomyces cerevisiae and the fungal pathogen Candida albicans possess cDNAs encoding for ferrioxamine permeases, a protein that allows the uptake of desferrioxamine (DFO)– Fe complexes (Yun et al., 2000; Hu et al., 2002). In the euphotic zone enhanced bioavailability of iron from Fe-organic complexes may result from photolysis of siderophores. Barbeau et al. (2001) demonstrated that when marine aquachelin–iron complexes are exposed to light Fe3q is reduced and the aquachelin molecules undergoes conformational change forming a lower affinity Fe3q ligand. There is enhanced iron bioavailability to eukaryote organisms via the increase in concentration of Fe2q. Experiments recently conducted by the authors showed that freshwater zebrafish iron uptake is significantly enhanced in the presence of the siderophore DFO, when DFO and FeCl3 is added in equal quantity to the water (the whole body control iron uptake was 2.26"0.19 pmol gy1 hy1 vs. DFO treated 4.12"0.62 pmol gy1 hy1; ns10 average"S.E.M.; P-0.05, student t-test). DFO is a hydroxamate siderophore produced by terrestrial organisms, but is often used as a model for marine siderophores with similar iron binding moieties, and Fe–DFO is bioavailable to marine prokaryotes (Hutchins et al., 1999). However, all experiments with zebrafish were performed in the light, and it is not clear whether the enhanced uptake is due to an increase in Fe2q concentrations following photolysis of DFO–Fe complex; or if DFO decreases intracellular iron concentrations which in turn results in an increase in the expression of proteins involved in iron uptake (Wardrop and Richardson, 1999); or if DFO simply retains more iron in solution increas-
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ing the possibility of interaction with the branchial apical membrane. 4. Molecular evidence for iron uptake in teleost fish cDNA sequences from a number of teleost fish species have been identified with high similarity to an evolutionary conserved group of membrane transport proteins termed solute carrier 11a1 (Slc 11a1) and solute carrier 11a2 (Slc 11a2), formerly known as natural resistance associated macrophage protein 1 (NRAMP1) and NRAMP2. These include cDNA clones from rainbow trout, Oncorhynchus mykiss, (Dorschner and Phillips, 1999), carp Cyprinus cyprinus (Saeij et al., 1999), sea bass, Morone saxatilis (GenBank accession number AY008746) and zebrafish, Brachydanio rerio (Donovan et al., 2002). Based on functional studies a number of Slc 11a proteins identified from diverse phyla (fungi, plants and mammals) have been linked with metal transport and more specifically ferrous iron transport (Gunshin et al., 1997; Eide, 1998; Rogers et al., 2000; Thomine et al., 2000; Cohen et al., 2000). The role of Slc 11a2 in mammalian intestinal iron uptake was identified in two separate laboratories via Xenopus oocyte expression cloning (Gunshin et al., 1997) and positional cloning to identify genes responsible for the microcytic anaemia in mice (Fleming et al., 1997). Functional studies revealed that the transporter was a Fe2q yHq symporter, operational in the pH range 5.5–7. The mRNA transcript is also found in most tissues, but predominantly in the small intestine or duodenum (Gunshin et al., 1997), which corresponds to the anatomical pattern of intestinal iron uptake, and is also the region where the intestine lumen fluids are slightly acidic. Other divalent essential metals such as Mn2q, Co2q, Cu2q, Zn2q, as well as the non-essential Cd2q and Pb2q could act as a substrate for the Fe2q yHq symporter (Gunshin et al., 1997), and due to this metal promiscuity it is often referred to as divalent metal transporter (DMT). Cd2q and Pb2q have also been shown to interfere with iron uptake into various cell lines (Tallkvist et al., 2001; Bannon et al., 2002), as well as interfere with iron entry into the enterocytes of the duodenum (Smith et al., 2002). In rainbow trout NRAMPyDMT transcripts are located in most tissues (Dorschner and Phillips, 1999) including the transport epithelia (N.R.B. personnel observa-
tion), suggesting that it may be involved in divalent cation uptake. A recent paper by Donovan et al. (2002) has demonstrated that a zebrafish homologue of DMT is capable of iron transport when expressed in Xenopus oocytes. In strategy I plants, yeast and mammals iron uptake via a Fe2q yHq symporter is linked to a membrane bound ferric reductase (Eide et al., 1992; Robinson et al., 1999; McKie et al., 2001). To date no teleost fish epithelial ferric reductase activity has been reported. In mammals this reductase is found in the duodenum and shares sequence homology to cytochrome b561 family of membrane reductases and is termed Dcytb (McKie et al., 2001). Whilst in ‘strategy 1’ plants, the membrane reductase is a ferric chelate reductase which utilises intracellular reducing power to liberate iron from its chelator and reduces Fe3q to Fe2q (Robinson et al., 1999; Weger et al., 2002). Three independent groups, one working on fish, simultaneously identified the protein responsible for basolateral membrane transfer of iron from cell to the blood and this transporter has been termed IREG1 (McKie et al., 2000), MTP1 (Abboud and Haille, 2000) or ferroportin (Donovan et al., 2000). Ferroportin was identified by positional cloning of the gene responsible for hypochromic anaemia in the zebrafish mutant weissherbst (Donovan et al., 2000). The similarity between ferroportin and IREG provides strong evidence that the machinery for cellular iron extrusion is evolutionarily conserved between fish and mammals. Ferroportin is located on the basolateral membrane of the enterocytes (McKie et al., 2000). The export of iron from the cell by this transporter depends on the presence of a membrane associated copper oxidase termed haephestin (Vulpe et al., 1995). Haephestin oxidises Fe2q as it leaves the cell resulting in Fe3q binding to transferrin. Transferrin is present in fish (Ford, 2001) and it is in this form that iron is transported to other tissues in the body. Based on molecular evidence the transport epithelial tissues of teleost fish possess the genes that encode for members of the Slc 11a family of proteins implicated in ferrous iron uptake, as well as for the transporter involved in basolateral membrane iron export. However, there is very little information on the functional characteristics of these genes, nor in fish, physiological data on the mechanisms of iron acquisition.
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5. Intestinal iron uptake in marine teleost fish Marine fish imbibe vast quantities of seawater to compensate for the osmotic loss of water across their integument. Water is absorbed along the whole length of the marine fish intestine, through NaCl coupled fluid absorption (Loretz, 1995). The movement of water from the lumen results in an increase in the concentrations of divalent cations as the fluid moves down the intestinal tract (Wilson, 1999; Grosell et al., 2001; Wilson et al., 2002). Concentrations of Ca2q and Mg2q can reach in excess of 10 and 100 mM, respectively which may in turn hinder the osmotic movement of water, but may at these concentration also be toxic. Perhaps, for this reason, marine fish intestine secrete large quantities of bicarbonate that causes a reduction of free Ca2q and Mg2q concentrations via the formation of Ca2q and Mg2q carbonates (Wilson, 1999; Wilson et al., 2002). These carbonates form a white precipitate that can be observed in most marine fish intestine lumens (Walsh et al., 1991). Even though bicarbonate secretion appears to play a key role in marine fish osmoregulation (Wilson, 1999; Wilson et al., 2002), it could potentially cause problems for intestinal divalent iron uptake. The presence of HCOy 3 at such high concentrations may limit the bioavailability of Fe2q, via the formation of Fe carbonates, and as a consequence of a large HCOy 3 secretion the lumen is alkaline (Wilson, 1999), which would result in a proton gradient incapable of providing the driving force for Fe2q uptake via a homologue of DMT i.e. a Fe2q yHq symporter. Despite such an adverse environment for ferrous iron transport we have recently shown in in vitro and in situ studies that the European flounder (Platichthys flesus) intestine absorbs iron (Bury et al., 2001). Iron uptake is significantly enhanced in the presence of the reducing agent ascorbate demonstrating that ferrous iron is more readily available compared to ferric iron (Bury et al., 2001). Flounder intestinal Fe2q uptake occurred predominantly in the posterior region and iron uptake in this region had a Q10 value of 1.94, compared to the Q10 value for iron uptake in the anterior and mid regions of the intestine being 1.54 and 1.16, respectively (Bury et al., 2001). Concomitantly, iron uptake in the posterior region showed an inverse linear relationship with haematocrit levels, and no such relationship was observed in the
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anterior or mid intestine (Bury et al., 2001). In mammals the anterior (duodenum) segment is the site for iron uptake, but our results suggest in marine fish iron crosses the intestine in the ferrous form in the posterior region. The reason for this disparity is not apparent and further studies to localise DMT transcript andyor protein in the intestine of teleost fish is required. How marine fish maintain intestinal Fe2q availability and accomplish uptake is unclear. However, it is hypothesised that several factors may all play a role, these include: epithelial mucus secretion resulting in metal-mucus chelates that retain the metal in solution in the intestinal tract (Glover and Hogstrand, 2002); reducing agents within the food such as ascorbate that increase the likelihood of Fe2q formation, and acidification of the boundary layer close to the tissue that also encourages Fe2q formation as well as enhancing the functioning of a proton symporter. In addition, it is tempting to speculate that a ferric reductase may be present on the brush border membrane of the marine fish intestine. 6. Branchial iron uptake in freshwater teleost fish To date, three studies have shown that the gill may be a site of iron uptake in freshwater fish (Roeder and Roeder, 1966; Andersen, 1997Bury and Grosell, unpublished data). Initially, Roeder and Roeder (1966) demonstrated that growth was perturbed in swordtail (Xiphodphorus helleri) and platyfish (Xiphodphorus helleri) fry reared in iron poor water and fed an iron-restricted diet. Reduced growth rate could be curtailed if FeSO4 (7.4 mg ly1, 134 mM) was added to the water, but not if iron was added as a ferric salt (Fe(NO3)3). This suggests that the iron needs of these juveniles could be met by uptake of iron from the water. What is not clear is why iron was preferentially taken up when added as a ferrous salt. Presumably Fe2q would be readily oxidised to the relatively unavailable Fe3q in a well oxygenated aquarium system, one can only surmise that present in the water were organic material that readily binds Fe2q and not Fe3q, thus increasing the concentration of dissolved iron available to the fish. Andersen (1997) exposed brown trout (Salmo trutta) late-eyed eggs, yolk-sac larvae or start-fed fry to 6.4 or 636 mmol (0.35 or 35 mg) Fe ly1, added as a combination of 59FeCl3 and ferric
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Fig. 1. Hypothetical representation of epithelial iron uptake in teleost fish. Briefly, ferric iron (Fe3q ) is reduced via a ferric reductase (FR). Ferrous iron (Fe2q) enters the cell via a Fe2qyHq symporter (DMT). Basolateral Fe2q export occurs via an iron-regulated transporter (FP), which is linked to a membrane bound copper containing oxidase, termed hephaestin (HP), that oxidises Fe2q to Fe3q. Fe3q binds to transferrin in the blood. It is uncertain whether there are also haem receptors (HR) in the intestine that mediate haem import; siderophore permeases (SP) in the gill that may aid siderophore bound iron uptake, or whether photolysis of Fesiderophores results in a greater abundance of Fe2q in the water for uptake by the fish. See text for more details. Abbreviations: Li, organic ligand (siderophore (sid), porphyrin, dissolved organic matter); DMT, divalent metal transporter; FR, Ferric reductase; HP, hephaestin; FP, ferroportin; HR, haem receptor; SP, siderophore permease; TF, transferrin.
ammonium citrate. Waterborne iron was unavailable to late-eyed eggs and yolk-sac larvae, suggesting that the developing embryos receive sufficient iron from the yolk. Interestingly, the ferroportin transcript has been located just below the membrane of the yolk cell of zebrafish (Donovan et al., 2000), suggesting that it is responsible for iron transport from the yolk to the embryo. The start-fed fry accumulated 59Fe from the water, but only at the higher concentration of 636 mmol Fe ly1. At this developmental stage the gills are forming and taking a more proactive role in ion acquisition from the water (Li et al., 1995). A recent study characterising the mechanism of branchial iron uptake by a freshwater teleost fish,
the zebrafish (Bury and Grosell, unpublished data) has shown that the kinetics of gill iron accumulation, when added to the water as FeCl3 has two components. At low iron concentrations (-40 nM) there is a saturable, high affinity component with an apparent Km of 5.9 nM Fe, and Vmax of 2.1 pmol Fe gy1 hy1. A linear component for iron uptake exists at higher iron concentrations (40– 200 nM). The apparent Km for the high affinity branchial iron transport is 25 and 325 lower than that for the high affinity iron uptake system of yeast (Eide et al., 1992), and for iron acquisition by Xenopus oocytes expressing DMT (Gunshin et al., 1997), respectively. The uptake system for siderophore–Fe complexes is the only other iron
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transport systems with such a high affinity for Fe (Winklemann, 2002), it is of interest to note that the siderophore DFO enhanced zebrafish iron uptake (see above). It is probable that the evolution of a branchial iron transport system with a remarkably low apparent Km is in response to the generally low dissolved iron concentrations in the aquatic environment. The addition of 2 mM of the reducing agent dithiothreitol (DTT) to the water, which enhances the formation of Fe2q (Cooper and Bury, personnel observation), significantly increased iron uptake into the gills at concentrations above 15 nM Fe. Bafilomycin, a proton pump inhibitor (Drose and Altendorf, 1997), reduced branchial iron uptake in the presence of DTT, suggesting that a proton gradient is required for iron uptake, and lends support to the idea that at low iron concentrations iron enters the gills via a Fe2q yHq symporter (Bury and Grosell, unpublished data). This is presumably via DMT, which has recently been cloned in zebrafish and has been shown to transports Fe2q in acidic (pH 6) environs (Donovan et al., 2002). However, it is unlikely that the pH of the boundary layer of the gill is this acidic (pH 6), and whether the DMT at the gill has different iron transport characteristic to its mammalian counterpart or is functioning sub-optimally awaits verification. Cadmium was the only of the divalent metals tested (Co2q, Ni2q, Pb2q, Cu2q, Zn2q, Cd2q and Mn2q) that significantly inhibited iron uptake into both the gills and body of zebrafish in the presence or absence of DTT. The calculated apparent iron– gill binding constant (log K) of 8.3 (based on the apparent Km for iron uptake) is similar to that for Cd binding to rainbow trout gills (Playle et al., 1993), and is at least an order of magnitude higher than the log K values for other gill–metals interactions (Playle, 1998; Bury and Hogstrand, 2002). However, the close coupling of the ferric reductase and DMT in other systems, and the competition between Cd2q in the absence and presence of DTT suggests that cadmium is interacting with iron at the Fe2q transport site. 7. Conclusion Based on the aquatic chemistry of iron it can be hypothesised that marine teleost fish obtain almost all of their daily requirements of iron from their diet. In the freshwater environment, there is
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the potential for the teleost fish to acquire iron from both the water as well as the diet. A recent study by the authors has shown that aquatic iron may well be a very important source of this nutrient to zebrafish. Indeed, there is now growing evidence that the aquatic uptake route for essential metals may contribute considerable to the overall maintenance of the organism metal homeostasis, particularly when the metal levels in the diet are low (Kamunde et al., 2002; Bury et al., 2003). Physiological evidence indicates that iron preferentially crosses the apical membrane of both the gills and intestine in the ferrous state (Bury et al., 2001. Molecular evidence would support this suggesting that the Fe2q yHq symporter (Dorschner and Phillips, 1999) belongs to the large Slc 11a family of evolutionary conserved proteins linked to Fe2q transport. However, functional analysis of these teleost Slc 11a genes is required. This symporter is probably linked to a reductase, which reduces either Fe3q or organic complexed iron to Fe2q prior to uptake (Fig. 1). However, other iron uptake pathways may be present, and iron bioavailability is enhanced if added to the diet of Atlantic salmon as haem compared iron salts (Andersen et al., 1997), suggesting the presence of a haem receptor mediated iron uptake process in the intestine (Crichton et al., 2002). References Abboud, S., Haille, D.J., 2000. A novel mammalian ironregulated protein involved in intracellular iron metabolism. J. Biol. Chem. 275, 19906–19912. Andersen, O., Dehli, A., Standal, H., Giskegjerde, T.A., Karstensen, R., Rorvik, K.A., 1995. Two ferritin subunits of Atlantic salmon (Salmo salar): cloning of the liver cDNAs and antibody preparation. Mol. Mar. Biol. Biotechnol. 4, 164–170. Andersen, O., 1997. Accumulation of waterborne iron and expression of ferritin and transferrin in early developmental stages of brown trout (Salmo trutta). Fish Physiol. Biochem. 16, 223–231. Andersen, F., Lorentzen, M., Waagbo, R., Maage, A., 1997. Bioavailability and interactions with other micronutrients of three dietary iron sources in Atlantic salmon, Salmo salar L. smolts. Aquacult. Nutr. 3, 239–246. Bannon, I., Portnoy, D., Olivi, M.E., Lees, P.S., Culotta, V.S., Bressler, J.P., 2002. Uptake of lead and iron by divalent metal transporter 1 in yeast and mammalian cells. Biochem. Biophys. Res. Commun. 295, 978–984. Barbeau, K., Rue, E.L., Bruland, K.W., Butler, A., 2001. Photochemical cycling of iron in the surface ocean mediated by microbial iron(III)-binding ligands. Nature 413, 409–413. Bianchini, A., Bowles, K.C., Brauner, C.J., Gorsuch, J.W., Kramer, J.R., Wood, C.M., 2002. Evaluation of the effects
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Grosell, M., Laliberte, C.N., Wood, S., Jensen, F.B., Wood, C.M., 2001. Intestinal HCOy 3 secretion in marine teleost fish: evidence for an apical rather than a basolateral Clyy HCOy 3 exchanger. Fish Physiol. Biochem. 24, 81–95. Guerniot, M.L., 2001. Improving rice yields-ironing out the details. Nature Biotechnol. 19, 417–418. Gunnars, A., Blomqvist, S., Johansson, D., Andersen, C., 2002. Formation of Fe(III) oxyhydroxide colloids in freshwater and brackish seawater, with incorporation of phosphate and calcium. Geochim. Cosmochim. Acta 66, 745–758. Gunshin, H., Mackenzie, B., Berger, U.V., et al., 1997. Cloning and characterization of a mammalian proton-coupled metalion transporter. Nature 388, 482–488. Hu, C.J., Bai, C., Zheng, X.D., Wang, Y.M., Wang, Y., 2002. Characterization and functional analysis of the siderophore– iron transporter CaArn1p in Candida albicans. J. Biol. Chem. 277, 30598–30605. Hutchins, D.A., Witter, A.E., Butler, A., Luther III, G.W., 1999. Differential utilization of chelated iron species by marine phytoplankton taxa. Nature 400, 858–861. Hyenstrand, P., Rydin, E., Gunnerhed, M., 1999. Response of pelagic cyanobacteria to iron additions—enclosure experiments from Lake Erken. J. Plankton Res. 22, 1113–1126. Jobling, M., 1995. Environmental Biology of Fishes. Chapman and Hall, London, UK. Jones, G.J., Pakenik, B.P., Morell, F.M., 1987. Trace metal reduction by phytoplankton: the role of the plasmalemma redox enzymes. J. Phycol. 23, 237–244. Kamunde, C., Grosell, M., Higgs, D., Wood, C.M., 2002. Copper metabolism in actively growing rainbow trout (Oncorhynchus mykiss): interactions between dietary and waterborne copper uptake. J. Exp. Biol. 205, 279–290. Li, J., Eygensteyn, J., Lock, R., et al., 1995. Branchial chloride cells in larvae and juveniles of freshwater tilapia Oreochromis mossambicus. J. Exp. Biol. 198, 2177–2184. Lienemann, C.P., Monnerat, M., Dominik, J., Perret, D., 1999. Identification of stoichiometric iron–phosphate colloids produced in a eutrophic lake. Aquat. Sci. 61, 133–149. Loretz, C.A., 1995. Electrophysiology of ion transport in the teleost intestinal cells. In: Wood, C.M., Shuttleworth, T.J. (Eds.), Cellular and Molecular Approaches to Fish Ionic Regulation, Fish Physiology, vol. 14. Academic Press, New York, pp. 25–56. Martin, J.H., Gordon, R.M., Fitzwater, S.E., 1991. The case for iron. Limnol. Oceanogr. 36, 1793–1802. McKie, A.T., Marciani, P., Rolfs, A., et al., 2000. A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol. Cell 5, 299–309. McKie, A.T., Barrow, D., Latunde-Dada, G.O., et al., 2001. An iron regulated ferric reductase associated with the absorption of dietary iron. Science 291, 1755–1759. Playle, R.C., Dixon, D.G., Burnison, K., 1993. Copper and Cadmium binding to fish gills—estimates of the metal-gill stability constants and modeling of metal accumulation. Can. J. Fish Aquat. Sci. 50, 2678–2687. Playle, R.C., 1998. Modelling metal interactions at fish gills. Sci. Total Environ. 219, 147–163. Robinson, N.J., Procter, C.M., Connolly, E.L., Guerniot, M.L., 1999. A ferric chelate reductase for iron uptake from soils. Nature 397, 694–697.
N. Bury, M. Grosell / Comparative Biochemistry and Physiology Part C 135 (2003) 97–105 Roeder, M., Roeder, R.H., 1966. Effect of iron on the growth rate of fishes. J. Nutr. 90, 86–90. Rogers, E.E., Eide, D.J., Guerinot, M.L., 2000. Altered selectivity in an Arabidopsis metal transporter. Proc. Natl. Acad. Sci. 97, 12356–12360. Rozan, T.F., Lassman, M.E., Ridge, D.P., Luther III, G.W., 2000. Evidence for iron, copper and zinc complexation as multinuclear sulphide clusters in oxic rivers. Nature 406, 879–882. Rue, E.L., Bruland, E.W., 1995. Complexation of iron(III) by natural organic-ligands in the central North Pacific as determined by a new competitive liquid equilibration adsorptive cathodic stripping voltametric method. Mar. Chem. 50, 117–138. Saeij, J.P.J., Wiegertjes, G.F., Stet, R.J.M., 1999. Identification and characterization of a fish natural resistance-associated macrophage protein (NRAMP) cDNA. Immunogenetics 50, 60–66. Salte, R., Rorvik, K.A., Reed, E., Nordberg, K., 1994. Winter ulcers of the skin in Atlantic salmon, Salmo salar L., pathogenesis and possible aetiology. J. Fish Dis. 17, 661–665. Smith, M.W., Shenoy, K.B., Debnam, E.S., Dashwood, M.R., Churchill, L.J., Srai, S.K., 2002. Divalent metal inhibition of non-haem iron uptake across the rat duodenal brush border membrane. Br. J. Nutr. 88, 51–56. Stumm, W., Morgan, J.J., 1996. Aquatic Chemistry. third ed. Wiley, Chichester, UK. Tallkvist, J., Bowlus, C.L., Lonnerdal, B., 2001. DMT1 gene expression and cadmium absorption in human absorptive enterocytes. Toxicol. Lett. 133, 171–177. Thomine, S., Wang, R., Ward, J.M., Crawford, N.M., Schroeder, J.I., 2000. Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes. Proc. Natl. Acad. Sci. 97, 4991–4996. Tipping, E., 1981. Adsorption by goethite (a-FeOOH) of humic substances from three different lakes. Chem. Geol. 33, 81–89. Vulpe, C.D., Kuo, Y.M., Murphy, T.L., et al., 1995. Hephaestin, a ceruloplasmin homologue implicated in intestinal iron
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Review
Iron acquisition by teleost fish Nicolas Burya,*, Martin Grosellb a
King’s College London, School of Health and Life Sciences, Franklin Wilkins Building, 150 Stamford Street, London SE1 9NN, UK b Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 33149-1098 Miami, FL, USA Received 31 October 2002; received in revised form 21 January 2003; accepted 21 January 2003
Abstract Iron is a vital micronutrient for teleost fish, being an integral component of proteins involved in cellular respiration and oxygen transfer. However, in excess iron is toxic, and fish need to balance uptake to prevent deficiency vs. potential toxicity. This review assesses the current physiological and molecular knowledge of the mechanisms of iron acquisition in the teleost fish. It focuses on freshwater teleost fish when assessing the gill as a possible site for iron acquisition, and includes a summary of geochemical processes that govern aquatic iron bioavailability. It focuses on marine teleost fish for assessing the mechanism of intestinal iron uptake. Physiological evidence indicates that iron preferentially crosses the apical membrane of both the gills and intestine in the ferrous (Fe2q) state. Molecular evidence supports this, demonstrating the presence of homologues in fish to the large Slc 11a family of evolutionary conserved proteins linked to Fe2q transport. This symporter is probably linked to a reductase, which reduces either ferric (Fe3q ) or organic complexed iron to Fe2q prior to uptake. 2003 Elsevier Science Inc. All rights reserved. Keywords: Divalent metal transporter; Ferroportin; Ferric reductase; Siderophore; Zebrafish; European flounder; Desferrioxamine; Metal transport; Copper
1. Introduction Iron is one of the most abundant metals on the earth and is essential to almost all organisms. This is because iron’s flexible redox activity enables it The present (N. Bury and M. Grosell: Iron acquisition by teleost fish) and subsequent paper (P. Chavez-Crooker, N. Garrido, P. Pozo, and G.A. Ahearn: Copper transport by lobster (Homarus americanus) hepatopancreatic lysosomes) originated from presentation given in the Homeostasis of essential yet toxic metals symposium, organizers Nic Bury and Martin Grosell, sponsored by the Society for Experimental Biology and the International Copper Association (ICA) at the American Physiological Society Conference – The Power of Comparative Physiology: Evolution, Integration and Application, San Diege, USA, 24–28th August 2002. *Corresponding author. Tel.: q44-2078484091; fax: q442078484500. E-mail address: [email protected] (N. Bury).
to be an integral component of cellular respiration, and in addition its positioning in haemoglobin enhances the oxygen carrying capacity of the blood. However, in excess iron can be toxic. At the cellular level iron catalyses the Fention reaction (Eq. (1)) resulting in the generation of free radical species, including hydroxyl radicals, that can potentially cause cellular oxidative damage. Fe2qqH2O2™Fe3qqOHyqOH•
(1)
Intracellular concentrations of iron are maintained stable via the regulation of iron import, export, as well as altering the cellular iron buffering capacity. The protein ferritin acts as a intracellular iron ‘soak’, taking up ferrous iron (Fe2q), oxidising Fe2q to ferric iron (Fe3q) and incorporating Fe3q into its molecule. Within the cell
1532-0456/03/$ - see front matter 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1532-0456Ž03.00021-8
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intracellular iron levels are monitored by iron regulatory proteins (IRP). In instances of reduced cellular iron concentrations the apo-IRP binds to iron regulatory elements (IRE) in the untranslated regions of the ferritin gene preventing translation; at times of excess iron the IRP is inactivated, and levels of ferritin increase (Crichton et al., 2002). cDNA encoding for the Atlantic salmon (Salmo salar) ferritin protein possess consensus IREs in the upstream untranslated region (Andersen et al., 1995), suggesting its involvement in the regulation of cellular iron concentrations. At the whole organism level there is no known regulated excretory pathway for iron; however, in vertebrates iron is lost via the bile as a consequence of haemoglobin breakdown and via sloughing of the intestinal epithelium. Consequently, iron homeostasis is solely maintained via the regulation of absorption from the diet. In fish there are two potential sites for metal uptake, across the intestinal (dietary borne) or branchial epithelium (water borne). To prevent metal deficiency or toxic excess fish need to judge their metal needs, assess the available levels of metal in the water or diet and then integrate regulation of branchial andyor intestinal import accordingly to meet demand. This critical minireview will focus on teleost fish iron requirements and the molecular and physiological evidence for potential mechanisms of iron acquisition at the branchial and intestinal epithelium. 2. Teleost fish iron requirements Iron is routinely added, as iron salts, to fish feeds in aquaculture, but the precise daily iron turnover rates for teleost fish are presently unknown. The concentrations of iron in the diet required to prevent signs of iron deficiency (i.e. perturbed haematological parameters) for salmonids are 60–100 mg Fe kgy1 (Andersen et al., 1997), channel catfish (Ictalurus punctatus) 30 mg Fe kgy1 (Gaitlin and Wilson, 1986), and puffer fish (Takifugu rubripes) 90–140 mg Fe kgy1 (Zibdeh et al., 2001). In the salmonid fish farming industry the levels of iron previously incorporated into the diets was considerably higher, but a reduction was implemented following evidence that excess iron may be responsible for the disease winter ulcers (Salte et al., 1994). A recent investigation estimated the daily loss of iron by the freshwater zebrafish to be 14 mg kgy1 dayy1
(Bury and Grosell, unpublished data), which compares favourably with the estimated loss of iron by humans (28 mg kgy1 dayy1 based on a loss of 1–2 mg Fe per day by a 70 kg male, Conrad et al., 1999). If we assume that fish eat approximately 2% of their body weight of food a day (Jobling, 1995), then the diet of a 1 kg fish must contain between 1.2 and 2 mg of iron a day. The uptake efficiency of iron from this diet required to balance iron loss would have to be in the order of 0.7– 1.2%. This is considerably lower than an uptake efficiency of between 5.5 and 16.6% for humans, based on the daily ingest rate of between 12 and 18 mg Fe (Crichton et al., 2002). The disparity may reflect the form in which iron is present in the feed. Andersen et al. (1997) showed that if iron is added as a haem complex then it is more readily bioavailable to Atlantic salmon than ferrous salts. It will be of interest to see if the daily iron requirements for commercially important species, such as the salmonids are similar to the zebrafish. Modifying the diet to include more readily bioavailable forms of iron will improve the quality of fish feeds for the aquaculture industry. 3. Bioavailability of aquatic iron The chemical form of iron (Fe3q, Fe2q, Feinorganic or -organic complexes), which the transport epithelium encounters will determine the mechanism of the apical membrane iron uptake pathway. (Reference to the intestinal chemistry will be presented in the section covered by Intestinal iron uptake in marine teleost fish). Fe2q is more readily available than Fe3q, but in oxic aquatic environments and at circumneutral pH Fe2q is readily oxidised to Fe3q. In addition, the bioavailability of iron to unicellular organisms in freshwater is far greater than that of seawater, and in the open oceans iron is the limiting nutrient of primary productivity (Martin et al., 1991). In freshwater the oxidation rate of Fe2q is considerably slower (hours) than in seawater (minutes) (Gunnars et al., 2002), and in these oxic environments iron is predominantly found as colloidal hydrous iron oxides that are not biologically available (Stumm and Morgan, 1996; Lienemann et al., 1999). The physio-chemical characteristics of seawater (high cation concentrations, alkaline pH) result in colloid aggregation (Forsgren et al., 1996), resulting in the half-time to removal of iron colloidal particles in seawater being considerably
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faster (;75 h), compared to freshwater (;1000 h) (Gunnars et al., 2002). This will in turn cause a slower turnover of iron in freshwater ecosystems. Fe(III) oxyhydroxide colloids, which may also be complexed with organic matter (Tipping, 1981), settle out and the reducing environs of the anoxic zone or sediment results in Fe(III) reduction and Fe(II) leaches from the sediment generating an iron cycle (Stumm and Morgan, 1996). In the summer months stratification of eutrophic lakes may result in an oxic zone extending only a few metres below the surface, and during the autumn the breakdown of the thermocline as the lake cools results in the mixing of the bottom nutrient and iron rich waters. Despite the propensity for iron oxidation in the freshwater environment it is not a limiting factor in lotic or lentic phytoplankton growth (Hyenstrand et al., 1999). Based on these observations it would appear that there is a greater potential for iron uptake by the gills of freshwater fish compared to marine fish, but that the diet is probably the most important source of iron in both environments. An important aspect of iron speciation in aquatic ecosystems, that has often been overlooked, is the presence of inorganic sulphides, which, despite being present at relatively low concentrations in oxic waters (-1 mM), form complexes with iron that may account for 20% of the total dissolved iron content (Rozan et al., 2000). At present there is no evidence that iron sulphide is bioavailable to fish. But, interestingly, silver when complexed to sulphide (Bowles et al., 2002) in the aquatic environment enhances silver bioavailability to the water flea (Daphnia magna) (Bianchini et al., 2002). The generally low concentrations of free iron in freshwater and marine environment has resulted in a number of organisms, including bacteria, yeast (Winklemann, 2002), and phytoplankton (Wilhelm and Trick, 1994; Weger et al., 2002), evolving specialised mechanisms for mobilising and sequestering iron. These organisms produce and secrete secondary metabolites, termed siderophores, with exceptionally high binding affinity (;log K 19) for iron (Witter et al., 2000). Siderophore production is not just restricted to the aquatic environment; strategy II plants also secrete them to aid iron uptake from the soil (Guerniot, 2001). Siderophores maintain iron in solution and these organisms have specialised membrane transport processes for the uptake of the iron–siderop-
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hore complex (Wilhelm and Trick, 1994; Winklemann, 2002; Cowart, 2002). It has been estimated that a large proportion of iron in solution, particularly in the open ocean, will be found as organic-Fe complexes (Wu and Luther, 1995). These will include the siderophore–Fe complexes, as well as Fe that originates from intracellular porphyrins, which are released upon cell death (Rue and Bruland, 1995; Hutchins et al., 1999). This organic chelated iron is available to marine eukaryote phytoplankton (Hutchins et al., 1999) probably via a membrane bound ferric reductase (Jones et al., 1987). However, other possible uptake routes of iron that are associated with organic matter may exist for other eukaryotes. The bakers yeast Saccharomyces cerevisiae and the fungal pathogen Candida albicans possess cDNAs encoding for ferrioxamine permeases, a protein that allows the uptake of desferrioxamine (DFO)– Fe complexes (Yun et al., 2000; Hu et al., 2002). In the euphotic zone enhanced bioavailability of iron from Fe-organic complexes may result from photolysis of siderophores. Barbeau et al. (2001) demonstrated that when marine aquachelin–iron complexes are exposed to light Fe3q is reduced and the aquachelin molecules undergoes conformational change forming a lower affinity Fe3q ligand. There is enhanced iron bioavailability to eukaryote organisms via the increase in concentration of Fe2q. Experiments recently conducted by the authors showed that freshwater zebrafish iron uptake is significantly enhanced in the presence of the siderophore DFO, when DFO and FeCl3 is added in equal quantity to the water (the whole body control iron uptake was 2.26"0.19 pmol gy1 hy1 vs. DFO treated 4.12"0.62 pmol gy1 hy1; ns10 average"S.E.M.; P-0.05, student t-test). DFO is a hydroxamate siderophore produced by terrestrial organisms, but is often used as a model for marine siderophores with similar iron binding moieties, and Fe–DFO is bioavailable to marine prokaryotes (Hutchins et al., 1999). However, all experiments with zebrafish were performed in the light, and it is not clear whether the enhanced uptake is due to an increase in Fe2q concentrations following photolysis of DFO–Fe complex; or if DFO decreases intracellular iron concentrations which in turn results in an increase in the expression of proteins involved in iron uptake (Wardrop and Richardson, 1999); or if DFO simply retains more iron in solution increas-
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ing the possibility of interaction with the branchial apical membrane. 4. Molecular evidence for iron uptake in teleost fish cDNA sequences from a number of teleost fish species have been identified with high similarity to an evolutionary conserved group of membrane transport proteins termed solute carrier 11a1 (Slc 11a1) and solute carrier 11a2 (Slc 11a2), formerly known as natural resistance associated macrophage protein 1 (NRAMP1) and NRAMP2. These include cDNA clones from rainbow trout, Oncorhynchus mykiss, (Dorschner and Phillips, 1999), carp Cyprinus cyprinus (Saeij et al., 1999), sea bass, Morone saxatilis (GenBank accession number AY008746) and zebrafish, Brachydanio rerio (Donovan et al., 2002). Based on functional studies a number of Slc 11a proteins identified from diverse phyla (fungi, plants and mammals) have been linked with metal transport and more specifically ferrous iron transport (Gunshin et al., 1997; Eide, 1998; Rogers et al., 2000; Thomine et al., 2000; Cohen et al., 2000). The role of Slc 11a2 in mammalian intestinal iron uptake was identified in two separate laboratories via Xenopus oocyte expression cloning (Gunshin et al., 1997) and positional cloning to identify genes responsible for the microcytic anaemia in mice (Fleming et al., 1997). Functional studies revealed that the transporter was a Fe2q yHq symporter, operational in the pH range 5.5–7. The mRNA transcript is also found in most tissues, but predominantly in the small intestine or duodenum (Gunshin et al., 1997), which corresponds to the anatomical pattern of intestinal iron uptake, and is also the region where the intestine lumen fluids are slightly acidic. Other divalent essential metals such as Mn2q, Co2q, Cu2q, Zn2q, as well as the non-essential Cd2q and Pb2q could act as a substrate for the Fe2q yHq symporter (Gunshin et al., 1997), and due to this metal promiscuity it is often referred to as divalent metal transporter (DMT). Cd2q and Pb2q have also been shown to interfere with iron uptake into various cell lines (Tallkvist et al., 2001; Bannon et al., 2002), as well as interfere with iron entry into the enterocytes of the duodenum (Smith et al., 2002). In rainbow trout NRAMPyDMT transcripts are located in most tissues (Dorschner and Phillips, 1999) including the transport epithelia (N.R.B. personnel observa-
tion), suggesting that it may be involved in divalent cation uptake. A recent paper by Donovan et al. (2002) has demonstrated that a zebrafish homologue of DMT is capable of iron transport when expressed in Xenopus oocytes. In strategy I plants, yeast and mammals iron uptake via a Fe2q yHq symporter is linked to a membrane bound ferric reductase (Eide et al., 1992; Robinson et al., 1999; McKie et al., 2001). To date no teleost fish epithelial ferric reductase activity has been reported. In mammals this reductase is found in the duodenum and shares sequence homology to cytochrome b561 family of membrane reductases and is termed Dcytb (McKie et al., 2001). Whilst in ‘strategy 1’ plants, the membrane reductase is a ferric chelate reductase which utilises intracellular reducing power to liberate iron from its chelator and reduces Fe3q to Fe2q (Robinson et al., 1999; Weger et al., 2002). Three independent groups, one working on fish, simultaneously identified the protein responsible for basolateral membrane transfer of iron from cell to the blood and this transporter has been termed IREG1 (McKie et al., 2000), MTP1 (Abboud and Haille, 2000) or ferroportin (Donovan et al., 2000). Ferroportin was identified by positional cloning of the gene responsible for hypochromic anaemia in the zebrafish mutant weissherbst (Donovan et al., 2000). The similarity between ferroportin and IREG provides strong evidence that the machinery for cellular iron extrusion is evolutionarily conserved between fish and mammals. Ferroportin is located on the basolateral membrane of the enterocytes (McKie et al., 2000). The export of iron from the cell by this transporter depends on the presence of a membrane associated copper oxidase termed haephestin (Vulpe et al., 1995). Haephestin oxidises Fe2q as it leaves the cell resulting in Fe3q binding to transferrin. Transferrin is present in fish (Ford, 2001) and it is in this form that iron is transported to other tissues in the body. Based on molecular evidence the transport epithelial tissues of teleost fish possess the genes that encode for members of the Slc 11a family of proteins implicated in ferrous iron uptake, as well as for the transporter involved in basolateral membrane iron export. However, there is very little information on the functional characteristics of these genes, nor in fish, physiological data on the mechanisms of iron acquisition.
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5. Intestinal iron uptake in marine teleost fish Marine fish imbibe vast quantities of seawater to compensate for the osmotic loss of water across their integument. Water is absorbed along the whole length of the marine fish intestine, through NaCl coupled fluid absorption (Loretz, 1995). The movement of water from the lumen results in an increase in the concentrations of divalent cations as the fluid moves down the intestinal tract (Wilson, 1999; Grosell et al., 2001; Wilson et al., 2002). Concentrations of Ca2q and Mg2q can reach in excess of 10 and 100 mM, respectively which may in turn hinder the osmotic movement of water, but may at these concentration also be toxic. Perhaps, for this reason, marine fish intestine secrete large quantities of bicarbonate that causes a reduction of free Ca2q and Mg2q concentrations via the formation of Ca2q and Mg2q carbonates (Wilson, 1999; Wilson et al., 2002). These carbonates form a white precipitate that can be observed in most marine fish intestine lumens (Walsh et al., 1991). Even though bicarbonate secretion appears to play a key role in marine fish osmoregulation (Wilson, 1999; Wilson et al., 2002), it could potentially cause problems for intestinal divalent iron uptake. The presence of HCOy 3 at such high concentrations may limit the bioavailability of Fe2q, via the formation of Fe carbonates, and as a consequence of a large HCOy 3 secretion the lumen is alkaline (Wilson, 1999), which would result in a proton gradient incapable of providing the driving force for Fe2q uptake via a homologue of DMT i.e. a Fe2q yHq symporter. Despite such an adverse environment for ferrous iron transport we have recently shown in in vitro and in situ studies that the European flounder (Platichthys flesus) intestine absorbs iron (Bury et al., 2001). Iron uptake is significantly enhanced in the presence of the reducing agent ascorbate demonstrating that ferrous iron is more readily available compared to ferric iron (Bury et al., 2001). Flounder intestinal Fe2q uptake occurred predominantly in the posterior region and iron uptake in this region had a Q10 value of 1.94, compared to the Q10 value for iron uptake in the anterior and mid regions of the intestine being 1.54 and 1.16, respectively (Bury et al., 2001). Concomitantly, iron uptake in the posterior region showed an inverse linear relationship with haematocrit levels, and no such relationship was observed in the
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anterior or mid intestine (Bury et al., 2001). In mammals the anterior (duodenum) segment is the site for iron uptake, but our results suggest in marine fish iron crosses the intestine in the ferrous form in the posterior region. The reason for this disparity is not apparent and further studies to localise DMT transcript andyor protein in the intestine of teleost fish is required. How marine fish maintain intestinal Fe2q availability and accomplish uptake is unclear. However, it is hypothesised that several factors may all play a role, these include: epithelial mucus secretion resulting in metal-mucus chelates that retain the metal in solution in the intestinal tract (Glover and Hogstrand, 2002); reducing agents within the food such as ascorbate that increase the likelihood of Fe2q formation, and acidification of the boundary layer close to the tissue that also encourages Fe2q formation as well as enhancing the functioning of a proton symporter. In addition, it is tempting to speculate that a ferric reductase may be present on the brush border membrane of the marine fish intestine. 6. Branchial iron uptake in freshwater teleost fish To date, three studies have shown that the gill may be a site of iron uptake in freshwater fish (Roeder and Roeder, 1966; Andersen, 1997Bury and Grosell, unpublished data). Initially, Roeder and Roeder (1966) demonstrated that growth was perturbed in swordtail (Xiphodphorus helleri) and platyfish (Xiphodphorus helleri) fry reared in iron poor water and fed an iron-restricted diet. Reduced growth rate could be curtailed if FeSO4 (7.4 mg ly1, 134 mM) was added to the water, but not if iron was added as a ferric salt (Fe(NO3)3). This suggests that the iron needs of these juveniles could be met by uptake of iron from the water. What is not clear is why iron was preferentially taken up when added as a ferrous salt. Presumably Fe2q would be readily oxidised to the relatively unavailable Fe3q in a well oxygenated aquarium system, one can only surmise that present in the water were organic material that readily binds Fe2q and not Fe3q, thus increasing the concentration of dissolved iron available to the fish. Andersen (1997) exposed brown trout (Salmo trutta) late-eyed eggs, yolk-sac larvae or start-fed fry to 6.4 or 636 mmol (0.35 or 35 mg) Fe ly1, added as a combination of 59FeCl3 and ferric
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Fig. 1. Hypothetical representation of epithelial iron uptake in teleost fish. Briefly, ferric iron (Fe3q ) is reduced via a ferric reductase (FR). Ferrous iron (Fe2q) enters the cell via a Fe2qyHq symporter (DMT). Basolateral Fe2q export occurs via an iron-regulated transporter (FP), which is linked to a membrane bound copper containing oxidase, termed hephaestin (HP), that oxidises Fe2q to Fe3q. Fe3q binds to transferrin in the blood. It is uncertain whether there are also haem receptors (HR) in the intestine that mediate haem import; siderophore permeases (SP) in the gill that may aid siderophore bound iron uptake, or whether photolysis of Fesiderophores results in a greater abundance of Fe2q in the water for uptake by the fish. See text for more details. Abbreviations: Li, organic ligand (siderophore (sid), porphyrin, dissolved organic matter); DMT, divalent metal transporter; FR, Ferric reductase; HP, hephaestin; FP, ferroportin; HR, haem receptor; SP, siderophore permease; TF, transferrin.
ammonium citrate. Waterborne iron was unavailable to late-eyed eggs and yolk-sac larvae, suggesting that the developing embryos receive sufficient iron from the yolk. Interestingly, the ferroportin transcript has been located just below the membrane of the yolk cell of zebrafish (Donovan et al., 2000), suggesting that it is responsible for iron transport from the yolk to the embryo. The start-fed fry accumulated 59Fe from the water, but only at the higher concentration of 636 mmol Fe ly1. At this developmental stage the gills are forming and taking a more proactive role in ion acquisition from the water (Li et al., 1995). A recent study characterising the mechanism of branchial iron uptake by a freshwater teleost fish,
the zebrafish (Bury and Grosell, unpublished data) has shown that the kinetics of gill iron accumulation, when added to the water as FeCl3 has two components. At low iron concentrations (-40 nM) there is a saturable, high affinity component with an apparent Km of 5.9 nM Fe, and Vmax of 2.1 pmol Fe gy1 hy1. A linear component for iron uptake exists at higher iron concentrations (40– 200 nM). The apparent Km for the high affinity branchial iron transport is 25 and 325 lower than that for the high affinity iron uptake system of yeast (Eide et al., 1992), and for iron acquisition by Xenopus oocytes expressing DMT (Gunshin et al., 1997), respectively. The uptake system for siderophore–Fe complexes is the only other iron
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transport systems with such a high affinity for Fe (Winklemann, 2002), it is of interest to note that the siderophore DFO enhanced zebrafish iron uptake (see above). It is probable that the evolution of a branchial iron transport system with a remarkably low apparent Km is in response to the generally low dissolved iron concentrations in the aquatic environment. The addition of 2 mM of the reducing agent dithiothreitol (DTT) to the water, which enhances the formation of Fe2q (Cooper and Bury, personnel observation), significantly increased iron uptake into the gills at concentrations above 15 nM Fe. Bafilomycin, a proton pump inhibitor (Drose and Altendorf, 1997), reduced branchial iron uptake in the presence of DTT, suggesting that a proton gradient is required for iron uptake, and lends support to the idea that at low iron concentrations iron enters the gills via a Fe2q yHq symporter (Bury and Grosell, unpublished data). This is presumably via DMT, which has recently been cloned in zebrafish and has been shown to transports Fe2q in acidic (pH 6) environs (Donovan et al., 2002). However, it is unlikely that the pH of the boundary layer of the gill is this acidic (pH 6), and whether the DMT at the gill has different iron transport characteristic to its mammalian counterpart or is functioning sub-optimally awaits verification. Cadmium was the only of the divalent metals tested (Co2q, Ni2q, Pb2q, Cu2q, Zn2q, Cd2q and Mn2q) that significantly inhibited iron uptake into both the gills and body of zebrafish in the presence or absence of DTT. The calculated apparent iron– gill binding constant (log K) of 8.3 (based on the apparent Km for iron uptake) is similar to that for Cd binding to rainbow trout gills (Playle et al., 1993), and is at least an order of magnitude higher than the log K values for other gill–metals interactions (Playle, 1998; Bury and Hogstrand, 2002). However, the close coupling of the ferric reductase and DMT in other systems, and the competition between Cd2q in the absence and presence of DTT suggests that cadmium is interacting with iron at the Fe2q transport site. 7. Conclusion Based on the aquatic chemistry of iron it can be hypothesised that marine teleost fish obtain almost all of their daily requirements of iron from their diet. In the freshwater environment, there is
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the potential for the teleost fish to acquire iron from both the water as well as the diet. A recent study by the authors has shown that aquatic iron may well be a very important source of this nutrient to zebrafish. Indeed, there is now growing evidence that the aquatic uptake route for essential metals may contribute considerable to the overall maintenance of the organism metal homeostasis, particularly when the metal levels in the diet are low (Kamunde et al., 2002; Bury et al., 2003). Physiological evidence indicates that iron preferentially crosses the apical membrane of both the gills and intestine in the ferrous state (Bury et al., 2001. Molecular evidence would support this suggesting that the Fe2q yHq symporter (Dorschner and Phillips, 1999) belongs to the large Slc 11a family of evolutionary conserved proteins linked to Fe2q transport. However, functional analysis of these teleost Slc 11a genes is required. This symporter is probably linked to a reductase, which reduces either Fe3q or organic complexed iron to Fe2q prior to uptake (Fig. 1). However, other iron uptake pathways may be present, and iron bioavailability is enhanced if added to the diet of Atlantic salmon as haem compared iron salts (Andersen et al., 1997), suggesting the presence of a haem receptor mediated iron uptake process in the intestine (Crichton et al., 2002). References Abboud, S., Haille, D.J., 2000. A novel mammalian ironregulated protein involved in intracellular iron metabolism. J. Biol. Chem. 275, 19906–19912. Andersen, O., Dehli, A., Standal, H., Giskegjerde, T.A., Karstensen, R., Rorvik, K.A., 1995. Two ferritin subunits of Atlantic salmon (Salmo salar): cloning of the liver cDNAs and antibody preparation. Mol. Mar. Biol. Biotechnol. 4, 164–170. Andersen, O., 1997. Accumulation of waterborne iron and expression of ferritin and transferrin in early developmental stages of brown trout (Salmo trutta). Fish Physiol. Biochem. 16, 223–231. Andersen, F., Lorentzen, M., Waagbo, R., Maage, A., 1997. Bioavailability and interactions with other micronutrients of three dietary iron sources in Atlantic salmon, Salmo salar L. smolts. Aquacult. Nutr. 3, 239–246. Bannon, I., Portnoy, D., Olivi, M.E., Lees, P.S., Culotta, V.S., Bressler, J.P., 2002. Uptake of lead and iron by divalent metal transporter 1 in yeast and mammalian cells. Biochem. Biophys. Res. Commun. 295, 978–984. Barbeau, K., Rue, E.L., Bruland, K.W., Butler, A., 2001. Photochemical cycling of iron in the surface ocean mediated by microbial iron(III)-binding ligands. Nature 413, 409–413. Bianchini, A., Bowles, K.C., Brauner, C.J., Gorsuch, J.W., Kramer, J.R., Wood, C.M., 2002. Evaluation of the effects
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