Waterborne iron acquisition by a freshwater teleost fish, zebrafish Danio rerio
1 Zoophysiological Laboratory, The August Krogh Institute, University of
Copenhagen, Denmark
2 King's College London, School of Health and Life Sciences, Franklin
Wilkins Building, 150 Stamford Street, London, SE1 9NN, UK
* Author for correspondence (e-mail: nic.bury{at}kcl.ac.uk)
Accepted 3 July 2003
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Summary |
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Key words: iron bioavailability, teleost, divalent metal transporter (DMT), ferric reductase, ferroportin, IREG, fish nutrition, metal, zebrafish, Danio rerio
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Introduction |
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Fish are unique amongst the vertebrates, because there is the potential for
nutritive metal acquisition from the water in addition to metal absorption
from the diet (Bury et al.,
2003). However, the ability of the gills to absorb iron will be
greatly influenced by geochemical factors that govern iron speciation. In oxic
conditions and at circumneutral pH, Fe(II) is readily oxidised and forms
colloidal hydrous iron oxides (Sturm and
Morgan, 1996
; Lienemann et
al., 1999
) that may be complexed with organic matter
(Tipping, 1981
). However, iron
has seldom been found to limit phytoplankton growth in freshwater lakes
(Hyenstrand et al., 1999
).
Many freshwater organisms have evolved mechanisms for mobilising and
sequestering iron. A number of freshwater bacteria, algae and cyanobacteria
produce organic compounds, siderophores, which are released to the environment
(Wilhelm and Trick, 1994
).
These siderophores have exceptionally high binding affinity
(
logK=19) for iron (Witter
et al., 2000
), thus maintaining iron in solution, and the iron
siderophore complexes are taken up
(Wilhelm and Trick, 1994
;
Cowart, 2002
). Some algal
species adopt a different mechanism, whereby a plasma membrane ferric chelate
reductase utilises intracellular reducing power to liberate iron from its
organic ligand and reduces Fe(III) to Fe(II), after which Fe(II) is
subsequently transported into the cells
(Robinson et al., 1999
;
Weger et al., 2002
). In
addition, photolysis of siderophore bound iron results in the formation of
lower affinity Fe(III) ligands and the reduction of Fe(III), increasing the
bioavailable Fe(II) fraction to organisms in the euphotic zone
(Barbeau et al., 2001
).
In the vertebrate small intestine, non-haem bound iron is reduced
via a membrane-bound ferric reductase
(McKie et al., 2001) and
ferrous iron enters the cell via a proton/Fe2+ symporter
(Gunshin et al., 1997
). This
latter protein belongs to the family of proteins termed natural resistance
associated macrophage proteins (Nramp), or solute carrier 11 type 2a (Sla 11
2a). However, the protein is more commonly known as the divalent metal
transporter (DMT), because it has been shown to transport other divalent
cations (Gunshin et al., 1997
;
Tallkvist et al., 2001
;
Bannon et al., 2002
). cDNA
clones with sequence similarity to members of the Sla112a family of proteins
have been identified in a number of fish species (see review by
Bury et al., 2003
). mRNA for
one of the rainbow trout Oncorhynchus mykiss Nramp isoforms has been
shown to be prevalent in the gills, intestine and kidney epithelia of
freshwater-adapted fish, suggesting its involvement in ion acquisition (C. A.
Cooper and N. R. Bury, personal observation). In zebrafish, iron leaves
intestinal cells via a protein termed ferroportin
(Donovan et al., 2000
).
Consequently, based on molecular evidence, the genes encoding proteins
involved in epithelial iron uptake appear to be present in teleost freshwater
fish.
Two studies have shown that the gill is potentially a site of iron uptake
(Roeder and Roeder, 1966;
Andersen, 1997
). Roeder and
Roeder (1966
) demonstrated
that growth was reduced in swordtail Xiphodphorus helleri reared in
iron-poor water and fed an iron-restricted diet. Growth rate was restored if
FeSO4 (7.4 mg l-1, 134 µmol l-1) was added
to the water. Andersen (1997
)
showed that start-fed brown trout larvae, a stage at which gills are formed
and the maternal source of nutrients the yolk-sac has been absorbed,
accumulate radiolabelled Fe when added as FeCl3 and at a
concentration of 35 mg l-1 Fe (627 µmol l-1 Fe) into
the water. At this developmental stage there is also an increase in whole body
expression of the iron binding protein transferrin
(Andersen, 1997
). To date,
there is no information on the mechanism of the branchial iron uptake.
Consequently, the present study characterises the iron uptake from the water
in freshwater zebrafish Danio rerio, and assesses the contribution
that this route of iron acquisition makes to whole body iron homeostasis.
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Materials and methods |
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Iron flux experiments
All iron uptake measurements were performed by assessing the accumulation
of radiolabeled 59Fe in the gills, body or whole body.
Radiolabelled ferric chloride (59FeCl3) dissolved in 0.5
mol 1-l HCl was obtained from Amersham Pharmacia Biotech (327 MBq
ml-1, 552 µg Fe ml-1). All radioactivity was counted
on a Canberra Packard Minaxi auto gamma 5000 series gamma-counter using the
manufacturer's guidelines.
All uptake experiments were run in daylight, in polyethylene bags containing 200 ml of deionised water at 28°C. Radioisotope was added 1 h before the addition of the fish, allowing for iron binding to the walls of the containers, which was approximately 50% of that originally added. Over the 4 h flux period applied in most experiments, counts in the water decreased on average by 13±1.6% (N=56), which equates to radioactivity taken up by the organism as well as that adsorbed to the surface of the polyethylene bags. To maintain constant pH an equal volume of 0.5 mol 1-l NaOH was added to the volume of 0.5 mol 1-l HCl added with the 59FeCl3. The final pH of the water was 6.34-6.78. A 1 ml water sample was taken for radioactive counting before the addition of 8-10 fish to the vessels. At the end of the flux period a second 1 ml water sample was taken and the fish underwent a wash protocol of 2x 1 min rinses in tapwater (total iron content 0.46 µmol l-1) and were then killed by an overdose of MS-222. The branchial basket was removed, and the masses of the gills and body after blotting dry with a paper towel recorded. This procedure removed radioactivity loosely bound to mucus and epithelia. Consequently, throughout this manuscript, branchial and body iron uptake refers to iron that has been transported into the cells or body, as well as that strongly bound to the epithelia following the wash protocol. Radioactivity present on the gills and in the body was measured separately. The time-course experiments were performed at a nominal iron concentration of 16.5±0.1 nmol l-1.
In the dose-dependency experiment, nominal iron concentrations in the water
below 50 nmol l-1 Fe consisted solely of radiolabelled
FeCl3, and desired concentrations higher than this were achieved by
addition of `cold' FeCl3. The `cold' FeCl3 was made by
dissolving FeCl3 in Milli Q (18 ) water on the day of the
experiment. To assess ferric versus ferrous iron uptake, zebrafish
iron influx studies were performed in the presence of 2 µmol l-1
of the reducing agent dithiothreitol (DTT). In a preliminary experiment the
proportion of 1 µmol l-1 FeCl3 in the ferric or
ferrous state following the addition of 3 µmol l-1 DTT was
49.5±5 and 50.5±5%, respectively (approximately 1:1), as
determined by the spectrophometric phenanthroline method
(Clesceri et al., 1998
). In
the absence of DTT the proportion of Fe3+ and Fe2+ was
81.3±6 and 18.8±6, respectively (approximately 4:1; C. A. Cooper
and N. R. Bury, unpublished data). Sodium influx measurements in the presence
of 2 µmol l-1 DTT were made in parallel to the iron flux
experiments to assess the general effect of reducing conditions on ion uptake.
All fluxes were performed in 200 ml of water ([Na+]=30 µmol
l-1) to which was added 0.1 µCi of 22Na+
(Dupont, Stockholm, Sweden; specific activity 11.2 MBq g-1 Na). 1
ml water samples were taken at the beginning and end of the experiment for
radioactive counting. At the end of the experiment fish were washed with
tapwater, killed with an excess of MS-222, and whole body radioactivity
recorded.
The influence of 50 nmol l-1 bafilomycin A (Sigma;
Vejlegårdsveg, Denmark), a V-type ATPase inhibitor
(Drose and Altendorf, 1997),
on whole body iron influx at 18 nmol l-1 59FeCl3 was
assessed in 10 fish over a 1 h exposure period and in a volume of 100 ml.
Bafilomycin A would be expected to reduce acidification of the gill
microenvironment by inhibiting H+ extrusion across the gills.
Metal competition studies in the presence or absence of 2 µmol
l-1 DTT were performed at 18.6±0.5 nmol l-1 Fe
(N=16), in the presence of 200 nmol l-1 each of the
following: CoCl2, NiCl2, PbNO3,
CuCl2, CdSO4, ZnCl2, MnCl2 or
FeCl3. All metal solutions were made by dissolving the appropriate
salt in Milli Q (18 ) water on the day of the experiment.
Iron depuration was assessed by pre-loading zebrafish with 59FeCl3 added to the water at a concentration of 21 nmol l-1 Fe for 24 h. After loading, 10 fish were taken, washed as described above and whole body radioactivity measured. The remaining fish were then placed in 10 litres of radioactive-free, deionised water at 28°C, and at 1, 7 and 28 days post-loading whole body radioactivity was determined. The fish were fed every other day during the depuration period and 50% of the water in the holding tank was replaced on alternate days. All calculations were decay corrected (59Fe t1/2=44.6 days) and there was no significance increase in fish mass over the 28-day period (t=0, 0.14±0.05 g; t=28, 0.15±0.06 g).
Calculations and statistics
Zebrafish iron or sodium uptake was determined from the following
equation:
Tissue Fe or Na+ uptake = c.p.m. / (SA x m x t), where c.p.m. is the counts min-1 in the tissue, SA is the specific activity of Fe (c.p.m. pmol-1) or Na (c.p.m. nmol-1) in the water, m (g) the mass of the fish, and t (h) the duration of the flux. For time-course experiments the time factor is removed.
A one-way analysis of variance (ANOVA) followed by a least-significance difference test was used to determine significant differences between gill or body iron uptake at different times during the time-course experiment. A Student's t-test was used to compare branchial, body or whole body iron uptake in control fish and those fish exposed to the various metals, or bafilomycin A. A Student's t-test was also used to compare whole body Na+ uptake in controls and those exposed to DTT. To assess the differences between the uptake rate in the presence and absence of DTT we compared the slopes of the linear regression lines for uptake above 40 nmol l-1. The `best-fit' lines to the data points were based on the regression coefficients as calculated by Sigma Plot 2001 computer graphics package.
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Results |
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The kinetics of gill iron accumulation in the absence of DTT (e.g. mostly Fe3+) showed two components. The first component, at concentrations below 40 nmol l-1 Fe, best fitted Michaelis-Menten kinetics with an apparent affinity Km=5.9±1.7 nmol l-1 Fe and Vmax=2.1±0.2 pmol g-1 h-1 (r2=0.96). Above concentrations of 40 nmol l-1 the uptake rate increased in a linear manner (y=0.83-0.7+0.0537±0.006x, r2=0.96) and did not saturate at 203 nmol l-1 Fe. In contrast, branchial gill accumulation of Fe in the presence of DTT (approximately 1:1 Fe3+:Fe2+) was linear over the whole range of Fe concentrations tested (y=0.15±0.3+0.13±0.001x, r2=0.99, Fig. 2A). Below water [Fe]=15 nmol l-1 there was no significant difference between the gill accumulation of iron in the absence or presence of DTT. But, based on the linear regression lines for iron uptake in the presence or absence of DTT at [Fe]>15 nmol l-1, uptake was significantly greater in the presence of DTT (P<0.05, Fig. 2A). That is, the higher [Fe2+] resulted in increased 59Fe uptake by the gills. Body Fe influx both in the presence and absence of DTT showed a sigmoidal pattern of uptake (Fig. 2B). DTT did not significantly affect Na influx (control, 226±30 µmol Na kg-1 h-1; in the presence of DTT, 155±14 µmol Na kg-1 h-1, P=0.067, Student t-test). Iron uptake of fish treated with 50 nmol l-1 bafilomycin A, in the presence of DTT, showed a significant decrease in whole body iron accumulation (control, 0.028±0.003 pmol g-1 h-1, N=10; versus bafilomycin A, 0.021±0.002 pmol g-1 h-1, N=10; P=0.02, Student t-test) (not shown).
|
Cadmium, at 200 nmol l-1, was the only metal tested to significantly reduce both gill and body Fe uptake in presence and absence of DTT (Fig. 3A,B). Of the other metals tested, only Mn(II) had a significant effect on Fe uptake, stimulating gill Fe accumulation in the presence of DTT (Fig. 3B).
|
Zebrafish loaded with 59Fe from the water then transferred to
radiolabel-`free' water lost 7.9 pmol Fe g-1 over the first 24 h
and then a further 5.7 pmol g-1 over the following 28 days
(Fig. 4). The kinetics of iron
depurations best fitted a two-component exponential model where
y=75.5(e-2.21t)+
10.2(e-0.0525t) and r2=1
(Sigma Plot 2001), and from which the radiotracer biological half-life for
each component can be calculated
(Bustamante et al., 2002). For
the short-lived component, t1/2=0.31 days, and the
long-lived component, t1/2=13.2 days.
|
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Discussion |
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The accumulation of Fe by zebrafish at exceedingly low concentrations
(16.5-21 nmol l-1), during the 4 h fluxes or the 24 h loading
period, contrasts with the observation that swim-up brown trout fry do not
accumulate iron over a 4-day exposure period to 6.4 µmol l-1
59Fe (Andersen, 1997). In
that study, brown trout fry only took up iron from the water at a higher iron
concentration of 640 µmol l-1
(Andersen, 1997
). This
disparity may reflect differences in the duration of the experiments, fish
species and/or water chemistries. In freshwaters, formation of ferric
oxyhydroxide occurs over a number of hours
(Gunnars et al., 2002
).
Consequently, in the present flux experiments the majority of the iron
probably remains in the dissociated Fe3+ state, whilst in longer
exposure periods (i.e. Andersen,
1997
) the iron may become unavailable. The quantity or nature of
the organic material or the sulphide concentration, which both form complexes
with iron (Rozan et al., 1999; Witter et
al., 2000
), were not measured in either study, but may also
influence iron bioavailability.
At [Fe]>40 nmol l-1 the linear, low-affinity component of
zebrafish branchial Fe uptake (in the absence of DTT) may represent a
non-specific uptake pathway. In contrast, the Michaelis-Menten kinetics of Fe
uptake at lower Fe concentrations (<40 nmol l-1) is indicative
of a physiologically regulated transport protein. This pattern of gill metal
uptake by freshwater fish has been observed for other metals
(Comhaire et al., 1994;
Hogstrand et al., 1996
;
Bury and Wood, 1999
;
Grosell and Wood, 2002
). For
example, copper uptake by rainbow trout shows saturation kinetics at low
ambient copper concentrations and a linear uptake component at higher copper
concentrations that are toxic (Grosell and
Wood, 2002
). The Km for the high-affinity
branchial iron transport is 25 times lower than that for iron uptake by the
bakers yeast Sacchoromyces cerevisiae
(Eide et al., 1992
), and 325
times lower than Xenopus oocytes expressing Nramp2/DMT
(Gunshin et al., 1997
). The
only other iron transport systems with affinity constants in the
nmol
l-1 region are for the uptake of siderophore-Fe complexes by
phytoplankton and the strategy II plants
(Granger and Price, 1999
;
Guerinot, 2001
). Evolution of
an iron transporter with a high affinity for iron is probably driven by the
freshwater Fe3+ concentration that the gill encounters.
The suggestion that it is the ferrous form (Fe2+) of iron that
enters the gills of fish is supported by the observation of Roeder and Roeder
(1966), who showed that the
reduced growth rate of swordtail fed an iron-poor diet and reared in ion-poor
waters could be prevented if ferrous, and not ferric, salts were added to the
water. Interestingly, intestinal uptake of iron by the European flounder
Platichthys flesus is predominantly in the ferrous form
(Bury et al., 2001
). Ferrous
iron is also the form in which iron is taken up by the strategy I plants
(Thomine et al., 2000
), some
yeast (Cohen et al., 2000
) and
mammals (Gunshin et al.,
1997
). To obtain ferrous iron from the environment these organisms
evolved a membrane ferric reductase associated with a ferrous iron transport
protein. In strategy I plants, the ferric chelate reductase is capable of
reducing ferric iron bound to organic matter in the soil
(Robinson et al., 1999
),
whilst in mammals the ferric reductase, termed Dcytb, shares similarities with
cytochrome b reductases (McKie et
al., 2001
). The enhanced uptake of iron in the presence of DTT at
[Fe]>15 nmol l-1, as well as the saturation kinetics exhibited
by this system, demonstrate that the apical membrane is the rate-limiting step
in iron uptake at low external iron concentrations, indicating the presence of
a branchial ferric (chelate) reductase. However, the similarity between uptake
in the absence and presence of DTT at [Fe]<15 nmol l-1 suggests
that the rate-limiting step is a combination of Fe3+ reduction and
Fe2+ entry.
The V-type ATPase inhibitor bafilomycin A reduced whole body Fe uptake.
Although an effect of apical membrane depolarization, caused by bafilomycin A,
on Fe uptake cannot be completely ruled out, these results suggests that the
Fe uptake is dependent on a proton gradient. A number of apical membrane
ferrous iron transporters have been identified that act as
Fe2+/H+ symporters
(Gunshin et al., 1997;
Andrews, 2000
).
Immunohistochemical evidence for a branchial apical membrane V-type ATPase has
been documented (Perry 1997
;
Wilson et al., 2000
). V-type
ATPases are responsible for the acidification of the water that passes over
the gill, and have been shown to govern zebrafish branchial Na+
uptake in ion-poor water (A. M. Z. Boisen and M. Grosell, unpublished data). A
partial cDNA sequence (accession number AF190508) with similarity to other
members of the Nramp family of proteins has been identified in the zebrafish,
and the proton pump has been localised in the zebrafish gill (A. M. Z. Boisen
and M. Grosell, unpublished data). Consequently, the likelihood is that the
branchial apical membrane ferrous iron uptake is via a divalent metal
transporter (DMT).
The similar pattern of concentration-dependent iron transfer, in the
presence or absence of DTT, from the gill into the body of zebrafish
(Fig. 2) suggest that iron,
whether in the ferric or ferrous form in the environment, is treated similarly
intracellularly. At high external iron concentrations the transport step from
the gill to the body becomes rate limiting and saturation of body iron uptake
is observed. The transporter involved in this process is probably ferroportin,
a basolateral ferrous iron transporter that has been identified in the
intestine of zebrafish (Donovan et al.,
2000). In other vertebrates, ferroportin is linked to a
copper-containing ferrioxidase termed hephaestin
(Vulpe et al., 1995
), which
oxidises Fe2+ so that iron circulates as Fe3+ bound to
transferrin.
Of the divalent metals tested, only Cd2+ inhibited iron uptake
into both the gills and body of zebrafish in the presence or absence of DTT
(Fig. 3). The lack of effect of
the metals, and indeed the stimulation by Mn(II), was a surprise because
Xenopus laevis oocytes injected with cRNA for the mammalian DMT show,
that in addition to Fe2+, they are capable of transporting other
divalent metals (Gunshin et al.,
1997). DMT homologues in the plant Arabidopsis thaliana
and the bakers yeast Saccharomyces cerevisiae also transport other
divalent metals (Thomine et al.,
2000
; Cohen et al.,
2000
). In addition, cadmium and lead both inhibit iron uptake by
various cell lines in culture (Tallkvist
et al., 2001
; Bannon et al.,
2002
), and lead interferes with iron uptake in the duodenum
(Smith et al., 2002
). The
close coupling of the ferric reductase and DMT in other systems indicates that
metals interact with iron at the Fe2+ transport site. However,
metals may also interact with Fe3+ or Fe2+ binding to
non-transport moieties on the apical surface. The calculated apparent gill
iron-binding constant (logK) of 8.3 (based on the apparent
Km for iron uptake) is similar to that for Cd binding to
fathead minnow gills (logK=8.6;
Playle et al., 1993
), and is
at least an order of magnitude higher than the logK values for other
gill-metal interactions (see table 2 of
Bury and Hogstrand, 2002
). The
competition between divalent Cd in the absence and presence of DTT shows that
it is directly interacting with iron following reduction, suggesting that
these metals share the same uptake route.
Depuration of radiolabelled iron following a loading from waterborne iron
follows a two-component exponential model (see
Fig. 4). The initial rapid
decline may represent loss of loosely bound iron from the gills and skin. The
degree to which this radiolabelled iron has been incorporated into `new'
proteins was not determined, but a loss rate of 0.53 pmol g-1
day-1 (i.e radiolabel lost over the whole depuration experiment)
equates to 28.4 µg kg-1 day-1, which is roughly
equivalent to the daily iron lost by humans, 14-28 µg kg-1
day-1 (1-2 mg Fe is lost by a 70 kg male per day;
Conrad et al., 1999). At the
lowest iron concentration tested (1.625 pmol), the body iron uptake rate met
this demand (0.47 pmol Fe g-1 h-1). However, it must be
stressed that the zebrafish in the present study were acclimated to deionised
water, where iron levels were exceptionally low (below detection limits),
which would be likely to result in an upregulation of branchial ion
transporters. This upregulation would lead to an overestimation of the uptake
rate from the gills when the water is supplemented with a metal or other ions.
However, support for the importance of iron uptake from the water in
contributing to iron homeostasis comes from the observations that growth rate
in swordtail can be enhanced if FeSO4 is added to the water
(Roeder and Roeder, 1966
).
In conclusion, we demonstrate that iron can be absorbed from the water in a
teleost fish, the zebrafish. The branchial iron transport shows
characteristics of other epithelial iron transport, coupling an apical
membrane ferric reductase to a ferrous transporter. The ferrous iron
transporter appears to be linked to a proton pump, suggesting that it probably
belongs to the large Scl 11 2a family of proteins involved in iron transport.
The affinity for this system is very high, with a Km=5.9
nmol l-1, which probably reflects the freshwater concentration of
iron available to teleost fish. Similar to other Fe2+/H+
symporters, it appears that the zebrafish branchial iron transporter is
inhibited by cadmium. No other divalent metals affected iron uptake at the
same concentrations, suggesting that this iron transport process is relatively
specific. The results obtained suggest that uptake of iron from the water may
be adequate to compensate for daily iron loss, which fits with recent results
demonstrating the contribution of branchial metal uptake in maintaining metal
homeostasis during times of metal deprivation
(Bury et al., 2003). For
example, Kamunde et al. (2002
)
demonstrated that rainbow trout fed a low copper diet (0.8 µg Cu
g-1) and reared in copper-poor water (0.4 µg Cu g-1)
showed reduced growth, which could be reversed by addition of low levels of
copper to the water. Based on radiotracer studies, 60% of the copper needs
were met from uptake via the water in the fish fed copper-depleted
diets (Kamunde et al., 2002
).
Rainbow trout thus have an integrative physiological mechanism whereby the
capacity for branchial metal uptake is upregulated to meet copper demand. In
the case of iron, it will be important to determine the relative proportion of
iron acquired from the diet or water in fish reared under different dietary or
waterborne iron regimes.
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Acknowledgments |
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Footnotes |
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References |
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