Dietary sodium inhibits aqueous copper uptake in rainbow trout (Oncorhynchus mykiss)
1 Dept of Biology, Nipissing University, North Bay, Ontario, P1B 8L7,
Canada
2 Dept of Biology, McMaster University, Hamilton, Ontario, L8S 4K1,
Canada
3 Dept of Zoology, University of Guelph, Guelph, Ontario, N1G 2W1,
Canada
* Author for correspondence (e-mail: gregp{at}nipissingu.ca)
Accepted 28 October 2002
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Summary |
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Key words: dietary sodium, aqueous copper uptake, fish, rainbow trout, Oncorhynchus mykiss
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Introduction |
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The primary mechanism of Cu toxicity to fish results from the combined
effects of a reduction in sodium (Na+) influx and an increase in
Na+ efflux, giving rise to a net reduction of plasma and whole body
Na+ (Laurén and McDonald,
1986,
1987b
;
Reid and McDonald, 1991
).
Reduced Na+ influx is thought to be associated with non-competitive
binding of Cu ions to the basolateral Na+-pump,
Na+/K+-ATPase, resulting in lower Na+ uptake
rates into the blood (Laurén and
McDonald, 1987a
; Pelgrom et
al., 1995
; Li et al.,
1996
,
1998
). Massive Na+
efflux is thought to be associated with Cu-induced damage to gill epithelia
that results in a reduction of the integrity of paracellular
`tight-junctions', rendering the epithelium more permeable to internal
Na+ (Laurén and
McDonald, 1986
; Evans,
1987
; McDonald and Wood,
1993
). The result of this net loss of Na+ is an
increase in blood viscosity and blood pressure, a compensatory tachycardia
and, under acutely toxic conditions, cardiac failure
(Wilson and Taylor, 1993
).
The etiology of waterborne Cu toxicity to freshwater fish, as described
above, is similar to that demonstrated in fish exposed to acidic water
(Milligan and Wood, 1982;
McDonald, 1983
;
McDonald and Prior, 1988
;
McDonald et al.,
1989a
,b
).
Under low pH conditions, fish demonstrate a similar ionoregulatory disturbance
that leads to a net reduction in whole body Na+. Sadler and Lynam
(1987
) first suggested that a
pH-induced ionoregulatory disturbance may be ameliorated by making use of
dietary ions. Studies by Dockray et al.
(1996
), Wilson et al.
(1996
) and D'Cruz et al.
(1998
) implicated an
ameliorative role of diet because satiation-fed fish exposed to acidic water
demonstrated little or no stereotypical ionoregulatory disturbances when
exposed to acidic water, contrary to findings in other studies where fish were
maintained on limited (or no) rations. Moreover, acid-exposed fish had greater
appetites relative to fish maintained under circumneutral conditions
(Dockray et al., 1996
). These
results prompted a subsequent study that identified dietary Na+
content, rather than dietary energy content, as the key component that reduced
ionoregulatory disturbances in acid-exposed fish
(D'Cruz and Wood, 1998
).
It seems from these studies that dietary Na+ plays some role in reducing stereotypical ionoregulatory disturbances in fish exposed to acidic water. The purpose of the present study was to determine if dietary Na+ plays a similar type of protective role in rainbow trout (Oncorhynchus mykiss) exposed to waterborne Cu and, if so, to understand the mechanism(s) involved. This was achieved by feeding fish increasing concentrations of dietary Na+ for one week, then challenging them with a short-term (6 h), sublethal exposure to waterborne Cu (20 µg l-1) to study the effect of dietary Na+ on Cu uptake, distribution and effect on ionoregulatory processes such as Na+ flux, Na+/K+-ATPase activity and drinking rates.
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Materials and methods |
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Experimental design
Four experiments were conducted during this study: (1) to determine the
effect of dietary Na+ on subsequent waterborne Cu uptake; (2) to
determine the effect of dietary Na+ on whole body Na+
concentrations and subsequent aqueous Na+ uptake; (3) to determine
the effect of dietary Na+ on the simultaneous appearance of newly
accumulated Cu and Na+ in the gill and on
Na+/K+-ATPase activity in gill tissue; and (4) to
determine the effect of feeding and dietary Na+ on drinking
rate.
In the first two experiments, five fish (mass 9-12 g) were randomly assigned to each of four 20-liter experimental tanks, where they were held for 7 days. Each of these tanks was supplied with approximately 100 ml min-1 of aerated, reconstituted soft water (see above). During the first 6 days of the 7-day exposure period, fish in each tank were fed at a rate of 3% total fish mass per day. Fish were not fed during the final 24 h. Fish in each tank received a single diet, where each diet ranged from 0.6% (control) to 3% Na+ by mass (see `Diet preparation' below). Virtually all of the food provided was eaten within the first few minutes. Uneaten food and feces were siphoned from each tank 20 min after feeding. This cleaning regimen, in addition to the flow-through experimental design, ensured that excess Na+ from Na+-supplemented diets did not accumulate in the water.
In these first two experiments, at the end of 7 days, fish were transferred for six hours to 6-liter plastic flux chambers that contained either 20 µg Cu l-1 in vigorously aerated, reconstituted soft water spiked with 55.5 MBq l-1 64Cu (first experiment) or reconstituted soft water spiked with 0.93 kBq l-1 22Na (second experiment; see `Copper and sodium fluxes' below). In neither case did the addition of radiotracer significantly change the Cu or Na+ concentrations in flux chambers. At the end of the 6 h flux in each experiment, fish were sacrificed by an overdose of MS-222 and dissected to separate tissues (see `Sampling and analysis' below).
In the third experiment, 72 fish (mass 60-105 g) were randomly assigned to two 150-liter polypropylene tanks (i.e. 36 fish per tank). One tank received a normal diet (i.e. 3% total fish mass per day, untreated trout food), while the other tank received a 3% Na+-supplemented diet at the same feeding rate. Fish were maintained under these conditions for seven days. On day eight (i.e. after a 24 h starvation period), 5-6 fish from each of the control and Na+-diet exposed groups were moved into 20-liter plastic containers to create four waterborne Cu and feeding treatments, namely `Fed', `Fed+Cu', `Na Fed' and `Na Fed+Cu'. Copper treatment comprised 20 µg l-1 labeled with 64Cu (55.5 MBq l-1, CuNO3) for 6 h. In addition, water for all the groups was spiked with 22Na (0.93 kBq l-1). Simultaneous exposure to 64Cu and 22Na allowed for the measurement of newly accumulated Cu and Na+ in the gill. Total Cu was also measured in gills. For each treatment group, in addition to the samples for radioisotope counting, a subsample of the gill (two middle gill arches) was dissected out and immediately frozen in liquid nitrogen for Na+/K+-ATPase activity analysis (see `Na+/K+-ATPase activities' below).
In the fourth experiment, drinking rates were determined in three groups of fish (mass 60-105 g), namely `Unfed control', `Fed control' and `Na-diet fed' (N=5-6) in the absence of waterborne Cu (see `Drinking rates' below). For this experiment, fish were moved into flux tanks a day before the experiment, and feeding took place in the flux tanks in the presence of the drinking rate marker ([3H]PEG-4000).
Diet preparation
All diets were prepared with granulated hatchery feed that had been ground
to a powder {Corey Feed Mills, Ltd; manufacturer's specifications: [Na]=0.3
mmol g-1 (6 mg g-1; i.e. 0.6%); [P]=0.4 mmol
g-1 (11 mg g-1); [Cu]=17.3 µg g-1; crude
protein=55%; crude fat=17%; crude fibre=2%}. Analytical grade NaCl was
dissolved in 40% v/w distilled, deionized water and mixed into a pre-weighed
sample of fish food to yield diets with 0.6% (control, no NaCl added but
subjected to the same treatment as other diets), 1.2%, 1.8% and 3%
Na+ by mass. The resulting paste was extruded through a pasta
maker, air-dried and broken into smaller pellets by hand. This method gave
Na+ concentrations that were very close to nominal values. Actual
measured Na+ concentrations in the four diets were: 0.25 mmol
g-1 (0.6%), 0.51 mmol g-1 (1.2%), 0.76 mmol
g-1 (1.8%) and 1.27 mmol g-1 (3%).
Copper and sodium fluxes
Fish were exposed to radioactive Na+ or Cu, as 22Na
(t1/2=31.2 months; Amersham Pharmacia Biotech, Inc.,
Piscataway, NJ, USA) or 64Cu (prepared at McMaster University
Nuclear Reactor from CuNO3, t1/2=12.65 h),
respectively. The use of radioisotopes allowed us to discriminate between
newly accumulated Na+ or Cu taken up by the fish during an
experimental exposure from Na+ or Cu already occurring in fish
tissues before exposure to elevated (experimental) dietary Na+ or
waterborne Cu concentrations. Consequently, specific radioactivity
corresponding to the Na+ or Cu isotope in fish tissues after
experimental exposures represents `newly accumulated' Na+ or Cu.
Newly accumulated Cu or Na+ was calculated by the following
equation (Grosell et al.,
1997):
![]() | (1) |
Unidirectional Na+ and Cu uptake rates were determined by summing the uptake into all the individual tissues and dividing the result by the specific radioactivity in the environment, the fish's mass (in kg) and the length of the exposure period (6 h) to convert to a rate. Net Na+ flux rates were calculated from the changes in total water Na+ over the flux period by analyzing water samples taken 15 min after the start of the flux and at the end of the 6 h flux period for Na+, as described in `Sampling and analysis' below. Sodium efflux was calculated from the difference between net Na+ flux and influx rates.
Sampling and analysis
In the first two experiments, gills, liver, kidney, gut [esophagus to
rectum, rinsed in deionized water (18 m Nanopure II, Sybron/Barnstead,
Boston, MA, USA) to remove any partially digested food], plasma and carcass
were dissected from fish. Gill sampling involved the removal of entire gill
baskets, because the volume of cartilaginous material was small and it was
impractical to separate it out in these juvenile fish. Whole blood was
collected by caudal puncture using 1 ml heparinized syringes fitted with
23-gauge needles. Blood samples were immediately centrifuged at 10 000
g for 5 min to separate cellular material from plasma. Separated
plasma was decanted from the cellular material and used in subsequent
analyses. 10 ml water samples were collected from each flux chamber [one at
the beginning (15 min) and the other at the end of the flux (6 h)] in all
three experiments and acidified with 100 µl concentrated HNO3
(trace metal grade, Fisher Scientific, Nepean, Ontario).
Whole body metal concentrations were calculated according to the following
equation:
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For Na+/K+-ATPase activities determined in the third experiment, only gill tissue was analyzed. Rather than entire gill baskets as in the previous two experiments, two middle gill arches per fish were used. Gill filaments were immediately frozen in liquid nitrogen for subsequent analyses.
Radioactivity in tissue and water samples containing 64Cu and 22Na was measured on a Canberra-Packard Minaxi Auto-Gamma 5000 series gamma counter with on-board automatic decay correction for 64Cu (Canberra-Packard Instruments, Meriden, CT, USA). In the third experiment, where fish were exposed to 64Cu and 22Na simultaneously, samples were counted immediately after dissection and were then stored for two weeks to allow the 64Cu to decay to undetectable levels. Samples were then recounted for 22Na and decay-corrected accordingly. The difference between the first and second count provided a measure of 64Cu activity. Tissue and water [3H]PEG-4000 activity was counted on a liquid scintillation counter (LKB Wallac 1217 Rackbeta; Pharmacia-LKB AB, Helsinki, Finland) using internal standardization.
After tissues (except plasma and water samples) were counted, they were digested in five volumes of 1 mol l-1 HNO3 (trace metals grade; Fisher Scientific) at 70°C for 24 h and subsequently centrifuged for 5 min at 10 000 g. A subsample of the supernatant (or whole plasma or water sample) was diluted appropriately in 0.5% HNO3. Total Cu concentrations were measured by graphite furnace atomic absorption spectrophotometry (GFAAS; Varian 1275 AA with GTA-95 atomizer; Mississauga, Ontario) using a 10 µl injection volume and operating conditions as suggested by the manufacturer for Cu. Total Na+ concentrations were measured using flame atomic absorption spectrophotometry (FAAS; Varian 1275). Certified analytical standards (National Research Council of Canada) analyzed simultaneously with experimental samples were within the specified range.
Na+/K+-ATPase activities
Na+/K+-ATPase activities were determined for two gill
arches from fish after 7 days of exposure to dietary Na followed by 6h of
acute exposure to waterborne Cu (20 µg l-1) using a slightly
modified version of the microplate UV detection method described in McCormick
(1993). Samples were frozen
immediately in liquid nitrogen and subsequently stored at -70°C until
analyzed for Na+/K+-ATPase activity. In this assay, the
rate of hydrolysis of ATP to ADP in the presence and absence of oubain (Sigma,
St Louis, MO, USA) was coupled to the oxidation of NADH to NAD+.
Changes in absorbance of the reaction mixture due to NADH oxidation were
measured at 340 nm over 15 s intervals for 10 min.
Na+/K+-ATPase activity was calculated as the difference
in ATP hydrolysis in the absence and presence of oubain and normalized to
total protein in each respective sample as determined by the Bradford
(1976
) method.
Drinking rates
Drinking rates were measured by the method of Wilson et al.
(1996) in unfed controls, fed
controls and in fish fed dietary Na+ for 7 days. Fish were exposed
to [3H]PEG-4000 at a concentration of 185 MBq l-1 in the
water for 6 h. This is less than the period of time (10 h) by which the tracer
reaches the anus at this temperature (C. M. Wood, unpublished results). Water
samples (10 ml) were taken 15 min after addition of [3H]PEG-4000
and again after the 6 h exposure. Fish were then killed with an overdose of
MS-222. The entire gastrointestinal tract was exposed by dissection, ligated
at the esophagus and rectum, removed and homogenized in five volumes of 8%
HClO3. Homogenate was processed for scintillation counting
according to Wilson et al.
(1996
), and a 1 ml sample was
counted. Plasma was also counted to ascertain that the [3H]PEG-4000
had been absorbed.
Drinking rate was calculated using the equation:
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Statistical treatment
All data are reported as means ± S.E.M. and were compared using
analysis of variance (ANOVA). In cases where data did not meet normality or
homogeneity of variance assumptions for ANOVA, significant differences were
determined using a nonparametric KruskallWallis rank sum test. Mean
Na+ and Cu concentrations in tissues of fish exposed to
Na+-supplemented diets in the first two experiments were compared
with those of fish fed the control diet (i.e. normal, untreated trout food)
using Dunnett's test. Mean Na+/K+-ATPase activities and
mean drinking rates were compared among experimental treatments using
TukeyKramer's honestly significant difference test. Mean differences
were considered to be significant when P<0.05.
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Results |
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Fish fed Na+-supplemented diets of 1.8% Na+ accumulated significantly less new Cu (as defined by equation 1) in a 6 h Cu flux period at 20 µg Cu l-1 than fish fed the control diet (0.6% Na+) in gill, liver and gut tissues (Fig. 2). New Cu uptake into kidney and plasma was reduced, but not significantly different from fish fed the control diet. Similarly, fish fed the 3% Na+ diet accumulated substantially and significantly less new Cu in gills, liver, kidney, plasma and gut relative to fish fed the control diet (in all cases P<0.05, N=4-5). Fish fed the 1.2% Na+ diet accumulated a similar amount of new Cu relative to those fed the control diet (P>0.05), showing that the threshold for effect lay between 1.2% and 1.8% dietary Na+.
|
Based on GFAAS analysis of total tissue Cu concentrations, only gill and liver tissues showed significantly lower total Cu concentrations in fish fed diets supplemented with 1.8% or 3% Na+ relative to those fed the control diet (Fig. 3). Over the 7 day exposure, fish fed the 3% Na+ diet exhibited a 25.1% reduction of Cu in the gills and a 44.5% reduction of Cu in the liver, relative to fish fed the control diet. In fish fed the 1.8% Na+ diet, only the 36.6% reduction in total liver Cu burden was significant. Neither kidney, plasma, gut nor carcass showed elevated total Cu relative to controls in any of the Na+-supplemented diet treatments (P>0.05).
|
Sodium uptake
After 7 days of the experimental feeding regime, total Na+
concentrations were significantly higher in gut tissue and plasma of fish fed
the 3% Na+ diet relative to fish fed the control diet
(Fig. 4). There were no
significant differences in other tissues or at lower dietary Na+
concentrations. As with waterborne Cu uptake rates, fish fed either 1.8% or 3%
Na+-supplemented diets demonstrated a significantly lower
waterborne Na+ uptake rate relative to fish maintained on the
control diet (Fig. 5;
P=0.02, N=4-5). Fish fed the 1.8% or 3%
Na+-supplemented diets took up waterborne Na+ 40.8% or
44.0% slower than fish fed the control diet. Waterborne Na+ and Cu
uptake rates were strongly and positively correlated with one another
(r=0.97, P=0.02, N=4-5). Na+ efflux
rates were also elevated in proportion to dietary Na+ load,
although these differences could not be evaluated statistically because they
were measured on whole treatment groups, not individuals. Branchial
Na+ efflux rates were -0.41 µmol g-1 h-1,
-0.45 µmol g-1 h-1, -0.47 µmol g-1
h-1 and -0.57 µmol g-1 h-1 in fish fed
control (0.6%), 1.2%, 1.8% and 3% Na+ diets, resulting in negative
net Na+ flux rates in the 1.8% and 3% Na+ treatment
groups.
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|
Na+/K+-ATPase activities
Na+/K+-ATPase activities in gill filaments
(Fig. 6) varied significantly
among experimental treatments. Generally, gills of fish exposed to waterborne
Cu had lower Na+/K+-ATPase activities than those that
were not exposed to Cu. Moreover, fish fed Na+-supplemented diets
showed higher gill filament Na+/K+-ATPase activity than
those fed regular diets. Consequently, fish that were fed a
Na+-supplemented diet and were exposed to waterborne Cu showed gill
Na+/K+-ATPase activity that was not significantly
different from control fish (i.e. Fed in
Fig. 6, representing normal
diet and no waterborne Cu).
|
Newly accumulated Na+ and Cu in the gills
Newly accumulated gill Na+ and Cu varied among experimental
treatments (Fig. 7). Gills of
fish from the Na Fed+Cu treatment accumulated less than one-third the amount
of new Na+ than those from the Fed+Cu treatment
(Fig. 7A; P<0.05,
N=5-6). However, new gill Na+ did not vary between fish
from Fed and Fed+Cu treatments, nor between Fed and Na Fed + Cu treatments.
Gills of Fed fish accumulated most new Cu, which was not significantly
different from those of Fed+Cu treatment (P>0.05) but was 59.6%
higher than in gills of fish from the Na Fed+Cu treatment
(Fig. 7B; P<0.05,
N=5-6).
|
Drinking rates
Mean drinking rates were low and never exceeded 2 ml kg-1
h-1 but still varied by treatment
(Fig. 8). Drinking rates were
not significantly different between fed and unfed fish (P>0.05).
However, they did significantly differ between fish fed a normal diet (i.e.
Fed) and those fed a 3% Na+-supplemented diet (P<0.05,
N=5). Fish fed the Na+-supplemented diet demonstrated the
highest drinking rates, which were 58.8% greater than in unfed fish.
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Discussion |
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Previous studies have shown that a dietary source of Na+ can be
just as important as a waterborne source for meeting physiological
requirements in rainbow trout (Smith et al.,
1989,
1995
;
D'Cruz and Wood, 1998
). Almost
100% of the Na+ taken up from the diet is absorbed though the gut
and taken up into the plasma (Smith et
al., 1995
). Fish can lose Na+ through their gills,
liver (via biliary excretion) and kidneys, although Na+
loss through the gills is much more important than other routes
(Smith et al., 1989
). Fish
maintain Na+ homeostasis by modulating influx and efflux, primarily
at the gills, as appropriate. Once plasma concentrations are elevated beyond
the needs of the fish, branchial Na+ efflux is stimulated and
influx is inhibited to ensure that electrolyte balance is maintained
(Salman and Eddy 1987
).
Fish can modulate branchial Na+ influx by changing the
activities of Na+/K+-ATPase in the basolateral membrane
and the proton pump, H+-ATPase, in the apical membrane
(McCormick, 1995;
Lin and Randall, 1995
;
Karnaky, 1997
).
Na+/K+-ATPase extrudes intracellular Na+ from
branchial epithelium into the blood, while H+-ATPase in the apical
membrane pumps protons out of the cell, which increases the electrochemical
gradient between the external and internal environments, thereby creating
conditions that favor waterborne Na+ influx
(Lin and Randall, 1995
).
Sodium efflux, on the other hand, is primarily diffusive and is modulated by
changes in internal concentration, in transepithelial potential (and therefore
in electrochemical gradient) and, most importantly, in gill permeability
(McDonald and Prior, 1988
;
McDonald et al., 1989b
). The
latter may reflect changes in both transcellular and paracellular
pathways.
Results from this study suggest that waterborne Cu uptake is strongly
associated with waterborne Na+ uptake and is therefore influenced
by the same ionoregulatory mechanisms that control Na+ homeostasis.
Our results reveal that branchial Na+ uptake was inhibited with
increasing dietary Na+ concentrations. This was apparent in the low
branchial Na+ uptake rates in fish fed diets containing 1.8% or 3%
Na+ (Fig. 5), which
corresponds well with other studies investigating the ionoregulatory effects
of dietary Na+ (Salman and
Eddy, 1987; Smith et al.,
1995
). Branchial Na+ uptake was probably inhibited in
these fish as a response to elevated plasma Na+ concentrations
(Fig. 4). At the same time,
branchial Cu uptake was also inhibited in fish fed 1.8% or 3% Na+
diets (Fig. 1). Moreover,
branchial uptake rates for waterborne Na+ and Cu were strongly and
positively correlated with one another (r=0.97, P=0.02).
Further evidence supporting a close relationship between aqueous
Na+ and Cu uptake is shown in
Fig. 7, where Cu-exposed fish
fed Na+-supplemented diets accumulated significantly less branchial
Na+ and Cu than those fed normal diets. Therefore, the evidence
collected in this study suggests that dietary Na+ inhibits
branchial Cu uptake. In fact, dietary Na+ was such an effective
branchial Cu uptake blocker that gills, livers, kidneys, plasma and guts of
fish fed Na+-enriched diets accumulated 50.0-88.2% less new Cu than
fish maintained on a normal diet (Fig.
2).
Our results were based on food treated with NaCl in order to increase
dietary Na+ concentrations. We cannot rule out the possibility that
reductions in branchial Cu uptake could be linked to dietary Cl-.
One possible way to determine if dietary Cl- plays a role in
reducing brachial Cu uptake is to repeat our experiments by supplementing food
with a different salt, such as NaSO4. However, given recent
evidence reported by Grosell and Wood
(2002) demonstrating a common
branchial Na+Cu uptake channel, we strongly suspect dietary
Na+, not Cl-, inhibits branchial Cu uptake.
Radiolabeled 64Cu was used in this study to distinguish between Cu taken up by fish during the 6 h exposure to elevated aqueous Cu (i.e. new Cu) and background Cu that occurred in tissues even before fish were exposed to elevated waterborne Cu (i.e. total Cu minus new Cu). An interesting effect of dietary Na+ in these fish was the significant reduction of total Cu in gills of fish fed 3% Na+-supplemented diets for the preceding 6 days, and in livers of fish fed either 1.8% or 3% Na+-supplemented diets (Fig. 3). Newly accumulated Cu in gills of fish fed the 3% Na+ diet accounted for only 5% of total gill Cu, whereas new Cu accounted for 0.11% and 0.04% of total Cu in livers of fish fed 1.8% and 3% Na+ diets, respectively. Therefore, new Cu accounted for only a small fraction of the total Cu load, especially in livers. The significantly lower total gill and liver Cu concentrations in fish fed high-Na+ diets suggest not only that normal Cu uptake was inhibited throughout the duration of the 6 day Na+-diet feeding period, even before fish were exposed to elevated waterborne Cu in the 6 h flux, but also that net Cu efflux may have been stimulated in the Na+-fed fish.
In addition to a reduction in branchial Na+ influx, fish fed
Na+-supplemented diets also demonstrated high Na+ efflux
in order to maintain electrolyte balance. Sodium efflux rates were 12% and 38%
higher in fish maintained on 1.8% and 3% Na+-supplemented diets,
respectively, resulting in negative net flux rates relative to fish maintained
on the control diet. This result corroborates other studies that have
demonstrated an increase in Na+ efflux in fish maintained on
Na+-supplemented diets (Smith
et al., 1995).
One of the ways that waterborne Cu causes ionoregulatory disturbances in
fish is via non-competitive inhibition of branchial
Na+/K+-ATPase activity
(Laurén and McDonald,
1987a; Sola et al.,
1995
; Pelgrom et al.,
1995
; Li et al.,
1998
). Intracellular Cu competes with Mg2+ for binding
sites on the ATP molecule and forms a physiologically inert ATP complex,
CuATP (Li et al.,
1996
). Normal functioning of Na+/K+-ATPase
requires MgATP. Once the basolateral
Na+/K+-ATPase is inhibited by Cu, the branchial cell can
no longer extrude intracellular Na+ into the blood (i.e. influx is
inhibited). Results reported here show Cu inhibition of branchial
Na+/K+-ATPase in both fed fish and Na+-fed
fish when exposed to waterborne Cu. However, in the Na+-fed fish,
the inhibition was not significant relative to the fed-fish control
(Fig. 6). This observation
suggests that in fish fed high-Na+ diets, Cu was less available in
branchial epithelium to inhibit Na+/K+-ATPase
activity.
Other studies examining the ionoregulatory effects of dietary
Na+, albeit at much higher concentrations (i.e. 12% Na+
w/w) than those used here, have demonstrated an increase in branchial
Na+/K+-ATPase activity
(Salman and Eddy, 1987), for
which there was a non-significant tendency in the present study. Increasing
Na+/K+-ATPase activity is commonly seen in
freshwater-adapted euryhaline fish after being transferred to saltwater
(McCormick, 1995
). The
increased Na+/K+-ATPase extrudes excess branchial
Na+ from the gills as a means of regulating Na+
homeostasis. Possibly, increased Na+/K+-ATPase activity
in gills of fish fed high-Na+ diets may serve to regulate branchial
Na+ in the same way.
Fish fed Na+-supplemented diets showed significantly higher
drinking rates than either fed or unfed fish
(Fig. 8). Undoubtedly, these
increased drinking rates represent another means by which Na+-fed
fish maintain osmoregulatory and ionoregulatory balance. Freshwater fish
usually drink only small quantities of water because of the hypotonic nature
of the dilute medium in which they reside, which causes a constant water
influx across the gills and body surface
(Karnaky, 1997). In order to
compensate for this water influx, fish produce a copious amount of dilute
urine and limit the amount of water they consume through drinking. However, as
internal Na+ concentrations increase with a high-Na+
diet, freshwater fish will increase their drinking rates to dilute the extra
Na+ absorbed from the diet.
It could be argued that increased drinking rates in Na+-fed fish might contribute to a higher waterborne Cu exposure through the gut, which could potentially offset any protection conferred by dietary Na+ observed in the gills. On average, Na+-fed fish drank 1.7 ml kg-1 h-1 of water that contained 20 µg Cu l-1 (Fig. 8). Therefore, these fish would consume approximately 0.034 ng g-1 h-1, which is at least 10-fold lower than Cu uptake rate across the gills (Fig. 1), so the contribution would be minor.
Taken together, results from this study suggest a common branchial uptake
route shared between waterborne Na+ and Cu
(Fig. 9). Recently, Grosell and
Wood (2002) have presented
evidence for two high-affinity mechanisms for branchial Cu uptake in the gills
of rainbow trout, one that directly competes for external Na+ and
another that is independent of external Na+. In accord with this
study, we speculate that this common route is probably in the form of an
apical Na+ channel, whose regulation depends on internal
Na+ concentrations, and a driving potential established by
H+-ATPase (Lin and Randall,
1995
). Dietary Na+ is taken up through the gut, causing
an increase in plasma Na+ concentrations. Plasma Na+ can
then be lost by passive diffusion to the water through the gills. Some of the
excess Na+ may be taken up by branchial cells via simple
diffusion or by a Ca2+/Na+-exchanger on the basolateral
membrane (Verbost et al.,
1994
). Sodium is also being taken up actively from the water
through a putative Na+ channel energized by the proton pump that it
shares with Cu. The putative Na+ channel would be downregulated in
response to elevated intracellular Na+ concentrations, resulting in
a sharp reduction of aqueous Na+ and Cu uptake. The net effect is
an increase in branchial Na+ efflux and a decrease in
Na+ and Cu influx.
|
Of course, the model we have proposed here does not preclude the other
(Na+-independent) branchial uptake routes for Cu characterized by
Grosell and Wood (2002).
Indeed, given that some new Cu accumulated in all tissues examined upon
exposure to waterborne Cu for 6 h, regardless of Na+ content in the
food, Cu was probably being taken up from the water by some other route in
addition to the shared Na+ channel as suggested here. In this
regard, Kamunde et al. (2001
,
2002
) have recently
demonstrated that branchial Cu uptake is also responsive to internal Cu status
of the fish. There is an interesting parallel here to cadmium metabolism,
which is thought to be taken up across the gill via apical
Ca2+ channels in the ionocyte
(Verbost et al., 1989
).
Recently, Zohouri et al.
(2001
) reported that elevated
dietary Ca2+ reduced but did not eliminate branchial cadmium uptake
in rainbow trout.
In conclusion, dietary Na+ effectively blocks waterborne Cu
uptake in rainbow trout. Aqueous Cu uptake inhibition was closely associated
with an inhibition of aqueous Na+ uptake. Consequently, we propose
that waterborne Na+ and Cu share a common apical channel that is
regulated, at least in part, based on internal Na+ requirements.
Although this study demonstrates the influence of dietary Na+ on
waterborne Cu uptake during short-term Cu exposures, the same principles seem
to apply under chronic exposure conditions
(Kamunde et al., in press).
Possible implications to wild fish may include protective effects of
high-Na+ diets against waterborne Cu toxicity in nature, active
dietary choice of high-Na+ food items by Cu-stressed fish, and
perhaps even Cu deficiency in fish raised on high-Na+ diets in
aquaculture. All these potential consequences deserve further
investigation.
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References |
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