Characterization of branchial lead-calcium interaction in the freshwater rainbow trout Oncorhynchus mykiss
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada
* Author for correspondence (e-mail: joerog{at}mcmaster.ca)
Accepted 8 December 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: waterborne lead, calcium, competition, rainbow trout, Oncorhynchus mykiss, branchial uptake
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent evidence has shown that the acute toxic mechanism for waterborne
lead in the rainbow trout Oncorhynchus mykiss is ionoregulatory
disruption, with observed effects on Na+ and Cl- balance
and Ca2+ homeostasis (Rogers et
al., 2003). These effects are manifested through an inhibition of
ion influx and corresponding decreases in the plasma levels of these ions.
This places lead midway between other known acute ionoregulatory toxicants
like copper and silver, which affect Na+ and Cl- balance
(Laurén and McDonald,
1985
; Wood et al.,
1996
; Morgan et al.,
1997
), and zinc and cadmium, which disrupt Ca2+
homeostasis (Spry and Wood,
1985
; Verbost et al.,
1987
).
Branchial uptake of Ca2+ is thought to be primarily by passive
movement through apical voltage-insensitive channels in the `chloride' cells
of the fish gill (Flik et al.,
1993). Once entering the chloride cell, Ca2+ is
transported via Ca2+-binding proteins to the basolateral
membrane where it is actively extruded into the circulation by way of a
high-affinity Ca2+-ATPase enzyme
(Flik et al., 1985
;
Verbost et al., 1994
;
Marshall 2002
) and/or a
Na+/Ca2+ exchange mechanism (Flik et al.,
1994
,
1997
;
Verbost et al., 1997
). Lead
could potentially have an impact at any one of these steps of calcium entry.
Recently, MacDonald et al.
(2002
) speculated that lead
disrupts Ca2+-homeostasis by competitive inhibition at apical
Ca2+ channels in the fish gill, thus entering the fish by the same
mechanism as Ca2+. There is an abundance of circumstantial evidence
in support of this. For example, the toxicity of waterborne lead is greatly
reduced with increasing water hardness, since the Ca2+ component
probably exerts protective effects by inhibiting entry of lead
(Sorensen, 1991
). Elevated
dietary Ca2+ levels have also been shown to reduce lead uptake in
fish (Varanasi and Gmur,
1978
). Once crossing the gill and entering the systemic
circulation, lead accumulates in bone, suggesting similarities between the
handling of lead and Ca2+ within the organism
(Davies et al., 1976
;
Holcombe et al., 1976
;
Settle and Patterson, 1980
).
These indirect relationships suggest that lead may share one or multiple
points of entry with calcium; however, despite such circumstantial evidence,
this relationship has not been proven directly.
Using both physiological and pharmacological techniques, metals like
cadmium and zinc have been shown to be Ca2+ antagonists. Both
metals have been found to reduce rates of Ca2+ influx
(Verbost et al., 1987;
Spry and Wood, 1985
) resulting
in hypocalcemia. Kinetic analyses of Ca2+ interaction with both
zinc (Spry and Wood, 1989
;
Hogstrand et al., 1994
,
1998
) and cadmium
(Niyogi and Wood, in press
)
have demonstrated typical Michaelis-Menten relationships, i.e. increased
Km values (decreased affinity of the apical calcium
channel) with little or no change in the maximal rate of uptake
(Jmax). This suggests direct competition. Additionally,
apical calcium channel blockers such as lanthanum have been shown to reduce
the rate of Ca2+, cadmium and zinc uptake, suggesting that these
metals share the same lanthanum-inhibitable route of uptake
(Verbost et al., 1989
;
Hogstrand et al., 1995
,
1996
). Using the rainbow trout
as a model species, the objective of the present study was to characterize the
branchial interaction of lead and Ca2+ by incorporating kinetic
analysis and the use of apical channel blockers (both voltage sensitive and
voltage insensitive). The potential stimulation of Ca2+-efflux, as
reported in lead-exposed brown trout
(Sayer et al., 1991
), and
possible inhibition of high-affinity Ca2+-ATPase were also
investigated as potential factors in lead-induced disruption of
Ca2+-homeostasis. Characterization of lead binding to the rainbow
trout gill may aid in the development of water chemistry-based predictive
models for lead, such as the Biotic Ligand Model (BLM;
Paquin et al., 2002
). This
process requires further understanding and characterization of key binding
sites involved in lead toxicity.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ca2+ influx measurements
Ca2+ influx determinations were carried out using methods almost
identical to those outlined by Rogers et al.
(2003), differing only in
lead-exposure concentrations used and duration of the pre-exposure period. In
brief, Ca2+ influx was measured by relating the specific activity
of 45Ca in the exposure water to the accumulation of isotope in the
fish following the flux period. In the present study, influx was measured
under control conditions and at lead concentrations that were approximately
50% and 25% of the 96 h LC50 of 4.8 µmol l-1
dissolved lead determined in City of Hamilton dechlorinated tapwater by Rogers
et al. (2003
). The nominal
lead-exposure concentrations implemented were 2.4 µmol l-1 and
1.2 µmol l-1. Juvenile rainbow trout were subject to pre-flux
exposure periods of 0 h, 12 h and 24 h before undergoing a flux period of 2 h
in control water or lead-containing water.
Kinetic analysis of the interaction between lead and Ca2+
The differential effects of lead on unidirectional calcium influx were
assessed in vivo using methods similar to those outlined in Zohouri
et al. (2001). Sixteen
polyethylene bags representing a series of four different calcium
concentrations in control and three lead concentrations, were filled with 3 l
calcium-free synthetic water (0.7 mmol l-1 Na+ and
Cl- added as NaCl, 1.9 mmol l-1 KHCO3, pH
8.0). Each bag was fitted with an airline and placed on a water bath for
temperature control (approximately 10°C). Three bags of each set were
spiked with a Pb(NO3)2 stock solution (Sigma; Aldrich,
Oakville, ON, Canada) to obtain nominal lead concentrations of 0.48, 2.4 and
4.8 µmol l-1 lead (control contained 0 µmol l-1
lead). Each bag was then spiked with Ca(NO3)2 (Fisher
Scientific, Markham, ON, Canada) to achieve nominal calcium concentrations of
150, 300, 600 and 1200 µmol l-1. Finally, flux bags were
injected with 7 µCi l-1 45Ca (as CaCl2,
specific activity 0.14 µCi mol-1; Perkin-Elmer, Boston, MA,
USA).
Juvenile rainbow trout were transferred to each of the 16 flux bags (seven fish per bag) and an initial 15 min period was allowed for `settling' and isotopic equilibration. The exposure period was 3 h; initial and final water samples (5 ml) were drawn in duplicate for determination of 45Ca activity and total calcium concentration. Water samples were also drawn for determination of total lead (unfiltered) and dissolved lead (filtered; 0.45 µm filter) concentrations. These samples were stored in plastic scintillation vials in 1% HNO3 for analysis. Following the 3 h flux period, fish were removed and killed by a single blow to the head. The fish were then rinsed for 1 min in 1 mmol l-1 EDTA (Sigma-Aldrich) followed by a 1 min rinse in a 5 mmol l-1 cold Ca2+ solution [Ca(NO3)2; Sigma-Aldrich] to remove all surface-bound 45Ca. Whole bodies were blotted dry, placed in scintillation vials, and digested in 1 mol l-1 HNO3 (Fisher Scientific; trace metal grade) at 55°C for 48 h. Samples were then homogenized by vortexing, a sample (1.5 ml) was removed, centrifuged at 13 000 g for 10 min, and the supernatant (1 ml) added to 5 ml of an acid-compatible scintillation cocktail (Ultima Gold; Packard Bioscience, Meriden, CT, USA). 45Ca radioactivity was measured by scintillation counting (Rackbeta 1217; LKB Wallac, Turku, Finland). Water samples taken for determination of 45Ca radioactivity were added to 10 ml of aqueous counting scintillant (ACSTM: Amersham, Piscataway, NJ, USA) and scintillation counted as above. 45Ca radioactivity was quench-corrected to the same counting efficiency as water samples by the method of external standard ratios, using a 45Ca quench curve generated from the tissue of interest in the same counting cocktail.
Water samples taken for the determination of total [Ca2+] were diluted with 0.2% lanthanum and analyzed by flame atomic absorption spectrophotometry (FAAS) using the Varian 220FS Spectr AA (Varian, Mulgrave, VC, Australia). Determination of total and dissolved waterborne lead concentrations was done using graphite furnace atomic absorption spectrophotometry (GFAAS; 220 SpectrAA) against a certified multi-element standard (Inorganic Ventures, Inc., Ventura, CA, USA).
The effect of calcium on branchial lead accumulation
The effect of waterborne calcium concentration on the branchial
accumulation of lead in juvenile rainbow trout was assessed using methods
similar to those implemented in the kinetic analysis of lead and
Ca2+, though precision was lower due to the lack of a suitable lead
radioisotope. Following a 3-h exposure period, experimental animals were
removed from the flux bags, killed by a blow to the head, and the gills
dissected. The gills were rinsed for 1 min in deionized water, blotted dry,
weighed and digested in 1 mol l-1 HNO3 at 55°C for
48 h. Samples were then homogenized by vortexing, a sample (approximately 1.5
ml) was removed, centrifuged at 13 000 g for 10 min, and the
supernatant analyzed for total lead concentration using GFAAS.
Calcium-channel blocker experiments
The role of apical calcium channels in mediating branchial lead uptake was
investigated by the use of the voltage-independent blocker, lanthanum,
antagonists of voltage-independent Ca2+ uptake, cadmium and zinc,
and the voltage-dependent channel blockers nifedipine and verapamil. In
assessing the effects of lanthanum on branchial lead accumulation, a series of
six polyethylene bags representing one control (0 µmol l-1
lanthanum) and five lanthanum concentrations were filled with 3 l of
synthetically modified water obtained by reverse osmosis (0.7 mmol
l-1 Na+ and Cl- added as NaCl), made
carbonate-free to reduce complexation of waterborne lanthanum
(Verbost et al., 1987;
Hogstrand et al., 1996
) and
calcium-free to maximize branchial lead accumulation. Flux bags were then
spiked with a LaCl3 (Sigma-Aldrich) stock solution to achieve
nominal waterborne lanthanum concentrations of 0.0001 to 1 µmol
l-1. Juvenile rainbow trout were transferred to each of the six
flux bags (seven fish per bag) and allowed a 10-min settling period for
equilibration with waterborne lanthanum. The bags were subsequently spiked
with a Pb(NO3)2 stock solution to achieve a nominal lead
concentration of 4.8 µmol l-1, followed by an additional
settling period to allow for lead equilibration. Water samples were taken,
filtered and unfiltered, for determination of total and dissolved lead
concentrations. The fish were then exposed for 3 h. Following exposure, fish
were removed from flux bags and the gills were dissected. Processing of the
dissected gills for total lead concentrations used procedures identical to
those outlined above. Using 45Ca as a radiotracer, simultaneous
measurements of Ca2+ influx were made at control and 1 µmol
l-1 lanthanum to assess the effect of lanthanum on Ca2+
uptake. Influx measurements were performed using a waterborne Ca2+
concentration of 600 µmol l-1. Further procedures used were
identical to those outlined above under `calcium influx measurements'. The
effects of cadmium and zinc on branchial lead accumulation and Ca2+
influx were assessed using methods almost identical to those used for
lanthanum. For cadmium, flux bags were spiked with a stock solution of
Cd(NO3)2 to achieve nominal concentrations of 0.0001,
0.001, 0.01, 0.1 and 1 µmol l-1 cadmium. For zinc, bags were
spiked with ZnSO4 to achieve nominal concentrations of 0.01, 0.1,
1, 10 and 100 µmol l-1 zinc. These experiments also included
measurements of gill cadmium burden from the same gill digests analyzed for
total lead, using GFAAS.
The use of nifedipine and verapamil as L-type calcium channel blockers was evaluated using methods almost identical to those used for lanthanum, cadmium and zinc with the exception of waterborne concentrations employed. For nifedipine, bags were spiked with a stock solution made using 75% ethanol (to solubilize the blocking agent; final ethanol concentration in the flux medium was 0.1%) to achieve concentrations of 0.1, 1, 10 and 100 µmol l-1 nifedipine. A control was run by spiking nifedipine-free water with ethanol to control for the effects of ethanol on lead accumulation and to maintain consistency between blocker experiments. For verapamil, flux bags were spiked with a stock solution made using deionized water (NANOpure II; Sybron/Barnstead, Boston, MA, USA) to achieve final concentrations of 0.1, 1, 10 and 100 µmol l-1 verapamil.
Effect of CaCl2 injection on Ca2+ influx and branchial lead accumulation
Methods used to test the effects of CaCl2 injection on
Ca2+ influx and branchial lead accumulation followed closely those
outlined in Hogstrand et al.
(1996). Ionic calcium was
injected into juvenile rainbow trout with the goal of reducing uptake of
Ca2+ and lead through the stanniocalcin-regulated pathway. In the
present study, fish were injected intraperitoneally with 0.22 µmol
l-1 Ca2+ g-1 body mass using an injection
solution made from CaCl2.H2O dissolved in 0.9% NaCl.
Control fish were sham-injected with 0.9% NaCl (vehicle only). Approximately
30 min following injection, measurements of Ca2+ influx were
performed on experimental and control fish (N=8 per treatment) using
methods described in the previous section. Branchial lead accumulation was
measured in a separate group of control and experimental fish (N=8
per treatment), again using methods outlined above.
Determination of Ca2+-efflux
The possibility of lead-induced stimulation of Ca2+-efflux was
investigated by following closely the methods of Perry and Flik
(1988). Fish were subject to
either no pre-flux exposure (control), or a pre-exposure period of 24 or 48 h
to nominal waterborne lead concentrations close to the 96 h LC50 of
4.8 µmol l-1. Lead exposure was carried out by dripping a stock
solution of Pb(NO3)2 (Sigma-Aldrich) dissolved in
deionized water (NANOpure II; Sybron/Barnstead, Boston, MA, USA) at a rate of
1 ml min-1 into a mixing tank fed with 500 ml min-1 of
City of Hamilton dechlorinated tapwater. The mixing tank then fed an exposure
tank (
200 l) holding 20 rainbow trout. At t=0 h, the exposure
tank was spiked with the lead stock solution to achieve the appropriate lead
concentration.
After the appropriate pre-exposure period, rainbow trout were anaesthetized
using MS-222 and given an intra-peritoneal injection of a 45Ca
solution (30 µCi 45Ca in 1 ml saline) in preparation for efflux
measurements. A period of 8-12 h was allowed for recovery of the fish, and for
isotopic equilibration. Fish were then transferred to darkened, Plexiglass
boxes (volume 450 ml), each fitted with an air supply. The boxes were filled
with either lead-free water or water spiked with
Pb(NO3)2 to achieve a nominal lead concentration of 4.8
µmol l-1, and placed in a water bath for temperature control.
Following a 0.5 h settling period, a 3 h Ca2+ flux measurement was
started. Initial water samples were taken in duplicate for 45Ca
activity and total calcium concentration (5 ml), and for determination of
dissolved lead concentrations (10 ml). At the end of the flux period,
comparable final water samples were drawn, fish were removed from their
respective exposure boxes and anaesthetized with MS-222. A terminal blood
sample was taken from each fish by caudal puncture, centrifuged at 13 000
g, and the plasma frozen for measurement of 45Ca
activity and total calcium concentration. Ca2+-efflux J
was calculated using the following formula:
![]() | (1) |
The effect of lead on high-affinity Ca2+-ATPase activity
High-affinity Ca2+-ATPase activity was assayed in adult rainbow
trout exposed to control conditions or to a nominal lead concentration of 4.8
µmol l-1 for 3 h, 24 h or 96 h in City of Hamilton dechlorinated
tapwater. Lead exposure was carried out using methods identical to those used
in measurements of Ca2+-efflux. Isolation of the basolateral
membrane from gill epithelium was carried out to assay enzyme activity.
Procedures followed closely those outlined in Perry and Flik
(1988). Trout were
anaesthetized with MS-222 and injected intravenously with 2500 U of sodium
heparin dissolved in 1 ml of saline. After 20 min, the fish were decapitated
just posterior to the pectoral fins. The head was placed onto an operating
table where the gills, irrigated with freshwater, were perfused with ice-cold
isotonic saline (0.6% NaCl) containing 20 U ml-1 of sodium heparin.
Perfusion was carried out at a pressure of 60 cmH2O via a
catheter (PE 60 tubing) inserted into the bulbus arteriosus until the gills
appeared to be free of trapped red blood cells. Following perfusion, the gill
basket was quickly excised. Further preparations were carried out on ice
(0-4°C) from this point.
The gill epithelium was scraped from the gill arches onto a glass plate using a glass microscope slide. The scrapings were then placed into a douncer with a loose pestle and homogenized in 15 ml of a hypotonic buffer consisting of 25 mmol l-1 NaCl and 20 mmol l-1 Hepes-Tris (pH 7.4). The volume was brought to 50 ml and the homogenate centrifuged for 15 min at 550 g (Beckman Ti 70 Rotor, Palo Alto, USA) to remove nuclei and cellular debris. Membranes were then collected by ultracentrifugation of the remaining supernatant at 30 000 g for 30 min. The resulting pellet, fixed to the centrifuge tube, was resuspended with 100 strokes of a douncer in 15 ml of an isotonic buffer containing 250 mmol l-1 sucrose, 12.5 mmol l-1 NaCl, 0.5 mmol l-1 H2EDTA, and 5 mmol l-1 Hepes-Tris (pH 7.5). The volume of the suspension was then brought to 30 ml with the same buffer. The resulting suspension was then centrifuged differentially in the following manner: 1000 g for 10 min, 10 000 g for 10 min, 30 000 g for 30 min. The resulting pellet was obtaining by adding 300 µl of a buffer containing 20 mmol l-1 Hepes-Tris (pH 7.4) and 200 mmol l-1 sucrose, and resuspended by passing the mixture through a 23-gauge syringe needle. The membrane preparations contained approximately 3 mg ml-1 of bovine serum albumin protein equivalents and were used on the same day of isolation. High-affinity Ca2+-ATPase activity was assayed in the presence of oligomycin B (5 µg ml-1) and 5 mmol l-1 sodium azide, at 1.0 µmol l-1 and 0 µmol l-1 free Ca2+ in an assay medium containing 100 mmol l-1 NaCl, 0.1 mmol l-1 ouabain, 0.5 mmol l-1 EGTA, 0.5 mmol l-1 N-hydroxyEDTA, 5 mmol l-1 MgCl2, 3 mmol l-1 Na2ATP and 20 mmol l-1 Hepes-Tris (pH 7.4). Membrane vesicles were permeabilized with 20 µg saponin mg-1 membrane protein. High-affinity Ca2+-ATPase activity was measured as the difference in inorganic phosphate release (Pi) in the presence and absence of Ca2+ in the assay medium.
The in vitro effect of lead on Ca2+-ATPase activity was tested by adding lead [from a Pb(NO3)2 stock solution] to the final membrane suspension, just prior to addition to the assay reaction medium, at concentrations similar to those measured in the final membrane suspension from the in vivo exposure. Finally, branchial lead accumulation was measured in a separate group of fish, similar in size and exposed to control conditions or to 4.8 µmol l-1 lead for 3 h, 24 h or 96 h.Procedures used for measurement of gill lead burden were identical to those outlined in the section `Calcium-channel blocker experiments' above.
Statistical analysis
In kinetic experiments, Michaelis-Menten analysis of the relationship
between the rate of Ca2+ influx
() and
waterborne calcium (substrate) were performed using Lineweaver-Burk linear
regression (double reciprocal plots), where Jmax (maximum
rate of influx) is the inverse of the y-intercept of the regression
line, and -Km (Michaelis constant) is the inverse of the
x-intercept of the regression line. For comparison, data were also
analysed by Eadie-Hofstee plots and by non-linear regression (Sigmaplot
2000).
Calculated data are expressed as means ± 1 S.E.M. (N). Experimental means were compared to corresponding control mean values by an unpaired two-tailed Student's t-test. Both time-dependent and dose-dependent responses in both control and experimental groups were tested against initial 0 h or control measurements using a one-way analysis of variance (ANOVA) with a two-sided Dunnett's post hoc multiple comparison. All statistical significance was calculated at P<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of lead on Ca2+ influx
Juvenile rainbow trout exposed to 2.3±0.1 µmol l-1
dissolved lead (measured) showed significant reductions in
Ca2+-influx rate compared to control fish
(Fig. 1A). Effects on influx
were immediate, with an approximate 40% inhibition occurring at 0-2 h of lead
exposure, this effect carrying through to 24-26 h, though non-significant at
12-14 h. Exposure to 1.4±0.2 µmol l-1 dissolved lead
resulted in similar inhibition of Ca2+ influx
(Fig. 1B), though significant
only at 12-14 h where the reduction was 32%. The concentration of total lead
in control water was 0.002±0.0001 µmol l-1.
|
Kinetic analysis of the interaction between lead and Ca2+
The saturable nature of calcium influx with increasing waterborne calcium
concentrations obeyed typical Michaelis-Menten kinetic analysis, in the
presence and absence of lead (Fig.
2). Measured waterborne lead concentrations were 0.46±0.03
µmol l-1, 2.5±0.2 µmol l-1 and
4.9±0.3 µmol l-1. Consistent with the immediate
inhibition of Ca2+-influx documented in the previous experiment,
waterborne lead exposure resulted in inhibition of calcium influx in juvenile
rainbow trout over a 3 h exposure period, the inhibition increasing with
increased lead concentrations. From analysis of a double reciprocal
Lineweaver-Burk plot (Fig. 3A),
it is apparent that Km values increased significantly with
increasing waterborne lead concentration
(Table 1),for example, 4.8
µmol l-1 lead exposure resulted in an approximate 16-fold
increase in Km value from control levels. By contrast,
Jmax was not significantly altered as a function of
waterborne lead concentration (Table
1). Kinetic analysis using Eadie-Hofstee and non-linear regression
also indicated significant increases in Km; however,
unlike the results obtained by Lineweaver-Burk regression, calculations
revealed a significant decrease (approximately 57%) in
Jmax, but only at the highest concentration, 4.8 µmol
l-1 lead (Table
1).
|
|
|
Because of the competitive nature of Ca2+-influx inhibition by
lead, an inhibitor constant was determined from a regression plot of apparent
Km (measured Km values from
Table
1)/Jmax vs. waterborne lead
concentration (Fig. 3B;
Segel, 1976). The
Ki,Pb (-Ki, x-intercept of
the regression line) was calculated to be 0.48 µmol l-1
lead.
The effect of calcium on branchial lead accumulation
The effect of waterborne calcium on branchial lead uptake is shown in
Fig. 4. Overall a protective
effect of calcium was observed, with the amount of branchial lead accumulation
decreasing with increasing waterborne Ca2+ concentration. At a
nominal waterborne lead concentration of 0.48 µmol l-1,
accumulation was consistent over the range of Ca2+ concentrations
(150-1300 µmol l-1). With increases in waterborne lead
concentrations to 2.4 (dissolved 2.5±0.2 µmol l-1) and
4.8 µmol l-1 (dissolved 4.9±0.3 µmol l-1),
the concentration of waterborne Ca2+ required to yield the same
protective effects increased. For example, upon exposure to 4.8 µmol
l-1 lead (dissolved 4.9±0.3 µmol l-1), lead
accumulation at 150 µmol l-1 Ca2+ was approximately
35 times higher than control (0 µmol l-1 lead) accumulation,
while at 1300 µmol l-1 Ca2+, accumulation was only 6
times greater than control gill lead accumulation. At a waterborne
Ca2+ concentration of approximately 1300 µmol l-1,
branchial lead accumulation measured after exposure to 0.48, 2.4 and 4.8
µmol l-1 lead did not differ significantly.
|
The effect of Ca2+ channel blockers on branchial lead accumulation
The effect of lanthanum, a voltage-independent Ca2+-channel
inhibitor, on Ca2+ uptake and branchial lead accumulation is shown
in Fig. 5A,B. From
Ca2+ influx measurements determined for control conditions at 600
µmol l-1 Ca2+ and 1 µmol l-1 lanthanum
(added as LaCl3), an approximate 55% reduction in Ca2+
uptake occurred in the presence of 1 µmol l-1 lanthanum. Upon
exposure to 2.3±0.2 µmol l-1 dissolved lead, waterborne
lanthanum significantly reduced gill lead accumulation in a dose-dependent
fashion. In the presence of 0.0001 µmol l-1 lanthanum, an
approximate 39% reduction in lead accumulation occurred compared to control
fish exposed to the same lead concentration in the absence of lanthanum. This
inhibition of lead uptake was dramatic from 0.001 to 1 µmol l-1
lanthanum, with a reduction of approximately 77% (compared to control) in lead
accumulation in the presence of these waterborne lanthanum concentrations.
|
Waterborne cadmium had similar effects on Ca2+ uptake and branchial lead accumulation. A waterborne concentration of 1 µmol l-1 cadmium reduced the rate of Ca2+ influx by approximately 74% compared to controls (Fig. 5A). Branchial cadmium accumulation occurred in a dose-dependent fashion when waterborne cadmium concentration was increased from control levels to 1 µmol l-1, with the largest increase occurring at the highest concentration (data not shown). This dose-dependent uptake corresponded with reduced levels of branchial lead accumulation (Fig. 5C). While gill cadmium accumulation increased by approximately 20-fold, lead uptake was reduced by 56% upon exposure to 2.3±0.2 µmol l-1 dissolved lead. Results using zinc as a calcium-channel blocker were similar. Ca2+ uptake was reduced by approximately 43% in the presence of 100 µmol l-1 zinc (Fig. 5A). This inhibition corresponded with a reduction of approximately 47% in branchial lead accumulation in fish exposed to 2.3±0.2 µmol l-1 dissolved lead in the presence of zinc (Fig. 5D). Similar to cadmium, zinc also accumulated dose-dependently at the gill, with the highest accumulation occurring at 100 µmol l-1 zinc (data not shown).
Voltage-dependent, L-type calcium-channel blockers did not appear to affect Ca2+ influx or branchial lead accumulation. Exposure to 100 µmol l-1 nifedipine or 100 µmol l-1 verapamil did not significantly reduce the rate of Ca2+ influx at 600 µmol l-1 Ca2+ (Fig. 6A). The effects of nifedipine on lead accumulation were found to be not significant compared to controls (Fig. 6B). Exposure to verapamil yielded similar results when comparing lead accumulation in the presence and absence of the blocking agent (Fig. 6C).
|
Effect of CaCl2 injection on Ca2+ influx and branchial lead accumulation
Injection of CaCl2 significantly reduced Ca2+ influx
by approximately 52% compared to fish sham-injected with vehicle only
(Fig. 7A). Inhibition of
Ca2+ influx corresponded with a significant decrease in branchial
lead accumulation in CaCl2 injected fish
(Fig. 7B). This reduction was
approximately 37% compared to controls (NaCl injected).
|
The role of efflux in lead-induced hypocalcemia
The role of efflux in lead-induced disruption of
Ca2+-homeostasis was assessed during exposure to control conditions
or 6.3±0.1 µmol l-1 dissolved lead. The results are
summarized in Table 2. Rainbow
trout exposed to control conditions (0 h) had an average
Ca2+-efflux
() of
approximately 32.8±4.6 nmol g-1 h-1. Lead-exposed
fish did not show significant deviations from this value after exposures of 3,
27 and 51 h to the experimental conditions.
|
High-affinity Ca2+-ATPase activity
Adult rainbow trout were exposed to a dissolved lead concentration of
5.5±0.4 µmol l-1. When compared to values for unexposed
fish at t=0 h (control sampling), activity after 3 h of lead exposure
was not reduced. However, prolonged exposure resulted in a reduction in
activity after 24 h, and a significant 80% inhibition by 96 h of lead exposure
(Fig. 8A). Branchial lead
accumulation increased significantly after 24 h and 96 h in fish exposed to an
identical waterborne lead concentration
(Fig. 8B). This accumulation
appeared to correlate well with the inhibition of high-affinity
Ca2+-ATPase activity (Fig.
8C). The in vitro effects of directly added
Pb(NO3)2 on the Ca2+-ATPase assay were
insignificant.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The observed rapid inhibition of calcium influx
(Fig. 1) after exposure to 1.4
and 2.3 µmol l-1 lead is consistent with previous studies
demonstrating reduced Ca2+ influx after exposure to lead
concentrations close to 4.8 µmol l-1
(Rogers et al., 2003). This is
contrary to findings presented by Sayer et al.
(1989
,
1991
) that lead exposure does
not result in disruption of Ca2+ influx. From the present data
(Figs 1 and
2), it is apparent that at
exposure concentrations (0.48 µmol l-1 upwards) that approach
environmentally relevant or normal levels (0.003-0.58 µmol l-1
lead; Demayo et al., 1982
), the
hypocalcemic effects of lead are still prominent. Additionally, influx
inhibition occurs immediately, suggesting that the interaction may be
competitive in nature, similar to metals like cadmium (Verbost et al.,
1987
,
1989
) and zinc
(Spry and Wood, 1985
;
Hogstrand et al., 1994
,
1996
). Therefore, measurements
of Ca2+ influx were useful for validating a kinetic approach to the
analysis of the relationship between lead and Ca2+.
The construction of uptake curves at a number of waterborne substrate
(Ca2+) concentrations using various waterborne lead levels allowed
for characterization of the effect of lead on branchial Ca2+ influx
kinetics. From these curves, Km and
Jmax values were calculated for each lead concentration
used (Table 1). These data
indicated a typical Michaelis-Menten competitive relationship with a
significant increase in Km value with increasing
waterborne lead concentrations. Increases in Km suggest
that as waterborne lead concentrations are elevated, the affinity of the
calcium-binding site for the calcium ion decreases, due to the presence of a
competitor. Consequently, the amount of Ca2+ required to achieve
half maximum transport is elevated. The stability of Jmax
values obtained from this kinetic study confirmed the competitive nature of
the lead/Ca2+ relationship, as the maximum rate of Ca2+
transport, or the integrity of Ca2+ binding sites, remained
unchanged. These findings are consistent with those made for zinc
(Hogstrand et al., 1994) and
for cadmium (Niyogi and Wood, in
press
), metals that are known calcium antagonists.
It is also important to note, however, that exposure to the highest lead concentration of 4.8 µmol l-1 may have caused a reduction in the maximum rate of Ca2+ influx. This was not detected by Lineweaver-Burk analysis, but was indicated by significant decreases in Jmax value when using Eadie-Hofstee and non-linear regression to interpret kinetic data. This would suggest that the branchial interaction between lead and Ca2+ might be described as a mixed competitive and non-competitive interaction upon exposure to elevated waterborne lead concentrations. This seems to be consistent with data presented in Fig. 2, that suggest the possibility of a Jmax decrease, Fig. 4, which suggests that exposure to 4.8 µmol l-1 waterborne lead results in a linear or diffusive component to lead influx when the waterborne concentration of Ca2+ is reduced (350 and 150 µmol l-1), and Fig. 8, which indicates that a high concentration of accumulated lead in the gill may inhibit baso-lateral high-affinity Ca2+-ATPase activity, albeit over a longer exposure period.
Kinetic analysis of lead and Ca2+ interaction allowed
calculation of an inhibitor constant (Ki,Pb) for calcium
influx of 0.48 µmol l-1. Based on this value, it is evident that
the affinity of lead for Ca2+ binding sites on the fish gill is
less than that for cadmium, but greater than that for zinc. Niyogi and Wood
(in press) reported an
inhibitor constant of 0.12 µmol l-1 for cadmium in rainbow
trout, a value 4 times lower than that reported in the present study for lead.
From the data of Hogstrand et al.
(1994
,
1998
), the inhibition constant
of zinc appears to be above 2 µmol l-1. This variation in
affinity for Ca2+ binding sites could explain the difference in
potency between lead, cadmium and zinc in terms of acute toxicity; cadmium is
the most toxic with a 96 h LC50 of 0.2 µmol l-1
(Hollis et al., 1999
) compared
to an LC50 of 4.8 µmol l-1 for lead
(Rogers et al., 2003
) and 13.3
µmol l-1 for zinc (Alsop et
al., 1999
). All values were determined in the same water
quality.
Branchial lead accumulation was largely dependent upon waterborne
Ca2+ concentrations, as shown in
Fig. 4. At the lowest
waterborne Ca2+ concentration of 150 µmol l-1, lead
accumulation significantly increased as waterborne lead concentrations
increased from control to 4.8 µmol l-1. This relationship
changed as Ca2+ concentration increased. Accumulation at 0.48, 2.4
and 4.8 µmol l-1 lead was not significantly different at higher
levels of waterborne Ca2+, demonstrating strong protective effects
of waterborne Ca2+ against branchial lead uptake. This confirms
other existing evidence highlighting the protective effects of water hardness
in lead toxicity (Sorensen,
1991) and suggests that it is the Ca2+ component of
hard water that dictates the toxicity of dissolved lead to fish. Richards and
Playle (1999
) reported similar
protective effects of Ca2+ against cadmium accumulation on the
gills in synthetically modified soft water. Alsop and Wood
(1999
) demonstrated reduced
zinc uptake and toxicity with increasing waterborne Ca2+
concentration. Considering the differential branchial lead accumulation and
kinetic lead/Ca2+ analysis from the present study, it is evident
that the gill is probably the major site of a predominately competitive
interaction between lead and Ca2+ that contributes to lead toxicity
in the rainbow trout.
Further evidence supporting the existence of a competitive relationship
between lead and Ca2+ was acquired through the use of apical
calcium-channel blockers. While inhibiting Ca2+ uptake
(Fig. 5A), waterborne
lanthanum, a classic Ca2+ channel blocker
(Weiss, 1974), significantly
reduced the amount of lead accumulation on the gill
(Fig. 5B). This suggests that
apical entry of lead into the chloride cells probably occurs through a
lanthanum-sensitive, voltage-independent apical calcium channel. These
observations are similar to those reported for zinc by Hogstrand et al.
(1996
), with an approximate
53% decrease in zinc uptake occurring in the presence of 1 µmol
l-1 lanthanum compared to controls. Comhaire et al.
(1998
) reported a 48%
inhibition of cobalt uptake at low-level lanthanum; however, 1 µmol
l-1 waterborne lanthanum exposure resulted in a stimulation of
cobalt influx. A similar effect was observed in the present study at
waterborne lanthanum concentrations exceeding 1 µmol l-1 (data
not shown). Stimulated lead accumulation may have been due to disruption of
apical membrane integrity by high lanthanum concentrations, resulting in an
increase in the diffusive component of lead uptake. Finally, the results of
the present study are consistent with those of Verbost et al.
(1987
), demonstrating an
inhibition of cadmium influx by lanthanum, and leading to the conclusion that
cadmium and Ca2+ share the same apical entry sites. This has led to
the use of cadmium for the purpose of calcium-channel antagonist experiments
(Comhaire et al., 1998
), as in
the present study.
The effects of cadmium on Ca2+ influx were significant
(Fig. 5A). As gill cadmium
accumulation increased with increased waterborne cadmium concentrations, gill
lead accumulation was reduced in a dose-dependent fashion compared to uptake
observed in the absence of the blocker
(Fig. 5C). This is consistent
with the results of Comhaire et al.
(1998), who reported
significant inhibition of cobalt uptake upon exposure to similar waterborne
cadmium concentrations. The inhibition of lead accumulation by cadmium, a
metal that traverses the chloride cell apical membrane through
voltage-independent, lanthanum-inhibitable channels
(Verbost et al., 1989
), is
further evidence supporting a similar entry route for lead. In further support
of this theory is the effect of zinc on lead accumulation
(Fig. 5D). The ability of zinc,
a metal believed to enter fish via Ca2+ uptake pathways
(Spry and Wood 1985
; Hogstrand
et al., 1994
,
1995
,
1996
,
1998
), to reduce branchial
lead accumulation suggests a similar route of entry for these metals.
The possibility of lead and Ca2+ transport through
voltage-dependent, L-type calcium channels at the apical surface of
the chloride cell was discounted by the lack of effect of the
voltage-dependent Ca2+ channel blockers verapamil and nifedipine
(Perry and Flik, 1988)
(Fig. 6A-C). Perry and Flik
(1988
) reported similar
results when assessing the effect of nifedipine on Ca2+ uptake,
leading to the suggestion that Ca2+ flux across the apical membrane
occurs through voltage-independent Ca2+ channels.
Injection of ionic Ca2+ was also employed to manipulate apical
Ca2+ channels so as to inhibit Ca2+ influx via
the stanniocalcin-controlled pathway. Previous studies have shown that
activation of this pathway results in a decreased permeability of chloride
cells to Ca2+ entry, thereby reducing influx
(Perry et al., 1989;
Verbost et al., 1993
). In the
present study, the hypocalcemic effects of stanniocalcin induction reduced
both Ca2+ influx and lead accumulation
(Fig. 7A,B). This provides
further evidence that lead uptake is by the same mechanism as Ca2+
uptake. These results are similar to those reported by Hogstrand et al.
(1996
), where both
Ca2+ and zinc influx rates were reduced by injection of
Ca2+.
The other significant process in the transepithelial uptake of
Ca2+ is the movement across the baso-lateral membrane against an
electrochemical gradient via a high affinity Ca2+-ATPase
mechanism (Perry and Flik,
1988). The importance of this enzyme to calcium transport makes it
a vulnerable target for Ca2+ antagonists such as cadmium, which,
following sufficient accumulation within the chloride cell, inhibits
Ca2+-ATPase activity contributing to disturbances in
Ca2+ homeostasis (Verbost et
al., 1989
). The present study revealed that prolonged lead
exposure results in significant inhibition of high affinity
Ca2+-ATPase activity (Fig.
8A). This reduction in activity occurred after 24-96 h of lead
exposure, suggesting that similar to cadmium, lead must accumulate in
sufficient amounts at the gill to have a negative effect on
Ca2+-ATPase activity (Fig.
8C). Accumulation at the gill was significantly elevated after 24
h and 96 h of lead exposure (Fig.
8B), the same time points where substantial inhibition of enzyme
activity was observed. This mechanism of Ca2+ homeostasis
disruption is also similar to that proposed for zinc
(Hogstrand et al., 1996
). With
increased amounts of branchial lead accumulation following prolonged exposure,
a slower, non-competitive component to disruption of Ca2+
homeostasis would occur. This would ultimately have an effect on
Jmax, decreasing the overall efficiency of the
Ca2+ transport system.
Ca2+ efflux was much less sensitive to lead than Ca2+
influx (Table 2). This is
evident by the absence of efflux stimulation at a waterborne lead
concentration (6.3 µmol l-1) that significantly reduced
Ca2+ influx (Rogers et al.,
2003) and is at least 4 times higher than the minimum
concentration required to elicit Ca2+ influx inhibition. These
findings are consistent with those for cadmium
(Verbost et al., 1987
;
Reid and McDonald, 1988
).
Verbost et al. (1987
) found
the effect of cadmium on Ca2+ efflux was insignificant, except for
measurements made at the highest cadmium concentration. Reid and McDonald
(1988
) found no effect of
cadmium on branchial Ca2+ efflux at circumneutral pH and at
moderately acidic pH (4.8). While significant stimulation of efflux by lead
was not observed, a slight, non-significant increase in efflux rate did occur
after 48 h of lead exposure. This could reflect a secondary effect of lead on
the integrity of paracellular routes, through which Ca2+ efflux is
thought to occur (Perry and Flik,
1988
).
Conclusions
To our knowledge, this study is the first to present direct evidence that
the uptake of waterborne lead by freshwater adapted rainbow trout is by the
same mechanism as Ca2+. The uptake involves competitive inhibition
of apical entry at lanthanum-sensitive Ca2+ channels and
interference with the function of the ATP-driven baso-lateral Ca2+
pump. Similarities are evident between lead and other known calcium
antagonists such as cadmium, zinc and cobalt, suggesting avenues for further
research that include characterization of the interaction between lead and
Ca2+-ATPase as well as the intracellular handling of lead by
Ca2+ transport proteins such as calmodulin.
This study could have potential implications for a predictive modelling
approach such as the Biotic Ligand Model (BLM)
(Paquin et al., 2002), which
is based on a toxic metal binding to the gills in competition with protective
and nutrient ions in the water column
(Playle et al., 1993
;
Playle, 1998
;
Paquin et al., 2002
).
Currently, binding models for lead are being developed
(MacDonald et al., 2002
), and
based on their predictions and the physiological evidence presented in this
study, it appears that lead is highly capable of out-competing Ca2+
for specific transport sites at the freshwater fish gill.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alsop, D. H., McGeer, J. C., McDonald, D. G. and Wood, C. M. (1999). The costs of chronic waterborne Zn exposure and the consequences of Zn acclimation on the gill/Zn interaction of rainbow trout in hard and soft water. Environ. Toxicol. Chem. 18,1014 -1025.
Alsop, D. H. and Wood, C. M. (1999). Influence of waterborne cations on zinc uptake and toxicity in rainbow trout, Oncorhynchus mykiss. Can. J. Fish. Aquat. Sci. 56,2112 -2119.[CrossRef]
Comhaire, S., Blust, R., Van Ginneken, L., Verbost, P. M. and Vanderborght, O. L. J. (1998). Branchial cobalt uptake in the carp, Cyprinus carpio: Effect of calcium channel blockers and calcium injection. Fish. Physiol. Biochem. 18, 1-13.[CrossRef]
Davies, P. H., Goettl, J. P., Sinley, J. R. and Smith, N. F. (1976). Acute and chronic toxicity of lead to rainbow trout Salmo gairdneri, in hard and soft water. Water Res. 10,199 -206.[CrossRef]
Demayo, A., Taylor, M. C., Taylor, K. W. and Hodson, P. V. (1982). Toxic effects of lead and lead compounds on human health, aquatic life, wildlife plants, and livestock. CRC Crit. Rev. Environ. Cont. 12,257 -305.
Flik, G., Kaneko, T., Greco, A. M., Li, J. and Fenwick, J. C. (1997). Sodium-dependent ion transporters in trout gills. Fish. Physiol. Biochem. 17,385 -396.[CrossRef]
Flik, G., Van Rijs, J. H. and Wendelaar-Bonga, S. E. (1985). Evidence for high affinity Ca2+-ATPase activity and ATP-driven Ca2+-transport in membrane preparations of the gill epithelium of the cichlid fish Oreochromis mossambicus. J. Exp. Biol. 119,335 -347.
Flik, G., Velden, J. A. V. D., Dechering, K. J., Verbost, P. M., Schoenmakers, T. J. M., Kolar, Z. I. and Bonga, S. E. W. (1993). Ca2+ and Mg2+ transport in gills and gut of tilapia, Oreochromis mossambicus: A review. J. Exp. Zool. 265,356 -365.
Flik, G., Verbost, P. M. and Atsma, W. (1994).
Calcium transport in gill plasma membranes of the crab Carcinus
maenus: Evidence for carriers driven by ATP and a Na+
gradient. J. Exp. Biol.
195,109
-122.
Hogstrand, C., Reid, S. D. and Wood, C. M. (1995). Ca2+ versus Zn2+ transport in the gills of freshwater rainbow trout and the cost of adaptation to waterborne Zn2+. J. Exp. Biol. 198,337 -348.[Medline]
Hogstrand, C., Verbost, P. M., Wendelaar-Bonga, S. E. and Wood, C. M. (1996). Mechanisms of zinc uptake in gills of freshwater rainbow trout: interplay with calcium transport. Am. J. Physiol. 270,R1141 -R1147.[Medline]
Hogstrand, C., Webb, N. and Wood, C. M. (1998).
Covariation in regulation of affinity for branchial zinc and calcium uptake in
freshwater rainbow trout. J. Exp. Biol.
201,1809
-1815.
Hogstrand, C., Wilson, R. W., Polgar, D. and Wood, C. M.
(1994). Effects of zinc on the kinetics of branchial calcium
uptake in freshwater rainbow trout during adaptation to waterborne zinc.
J. Exp. Biol. 186,55
-73.
Holcombe, G. W., Benoit, D. A., Leonard, E. N. and McKim, J. M. (1976). Long-term effects of lead exposure on three generations of brook trout (Salvelinus fontinalis). J. Fish. Res. Bd. Can. 33,1731 -1741.
Hollis, L., McGeer, J. C., McDonald, J. G. and Wood, C. M. (1999). Cadmium accumulation, gill cadmium binding, acclimation, and physiological effects during long term sublethal cadmium exposure in rainbow trout. Aquat. Toxicol. 46,101 -119.[CrossRef]
Laurén, D. J. and McDonald, D. G. (1985). Effects of copper on branchial ionoregulation in the rainbow trout (Salmo gairdneri) Richardson. J. Comp. Physiol. 155,635 -644.
MacDonald, A., Silk, L., Schwartz, M. and Playle, R. C. (2002). A lead-gill binding model to predict acute lead toxicity to rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 133C,227 -242.
Marshall, W. (2002). Na+, Cl-, Ca2+ and Zn2+ transport by fish gills: retrospective review and prospective synthesis. J. Exp. Biol. 293,264 -283.
Morgan, I. J., Henry, R. P. and Wood, C. M. (1997). The mechanism of silver nitrate toxicity in freshwater rainbow trout (Oncorhynchus mykiss) is inhibition of gill Na+ and Cl- transport. Aquat. Toxicol. 38,145 -163.[CrossRef]
Niyogi, S. and Wood, C. M. (in press). Comparative analyses of branchial calcium and cadmium transport kinetics and their interactions between rainbow trout and yellow perch, two species differing greatly in acute waterborne cadmium sensitivity. J. Comp. Physiol.
Paquin, P. R., Gorsuch, J. W., Apte, S., Batley, G. E., Bowles, K. C., Campbell, P. G. C., Delos, C. G., Di Toro, D. M., Dwyer, R. L., Galvez, F. et al. (2002). The biotic ligand model: a historical overview. Comp. Biochem. Physiol. 133C, 3-35.
Perry, S. F. and Flik, G. (1988). Characterization of branchial transepithelial calcium fluxes in freshwater rainbow trout, Salmo gairdneri. Am. J. Physiol 23,R491 -R498.
Perry, S. F., Seguin, D., Lafeber, F. P. J. G., Wendelaar-Bonga, S. E. and Fenwick, J. C. (1989). Depression of whole body calcium uptake during acute hypercalcemia in American eel, Anguilla rostrata, is mediated exclusively by corpuscles of Stannius. J. Exp. Biol. 147,249 -261.
Playle, R. C. (1998). Modelling metal interactions at fish gills. Sci. Total. Environ. 219,147 -163.[CrossRef]
Playle, R. C., Dixon, D. G. and Burnison, K. (1993). Copper and cadmium binding to fish gills: estimates of metal-gill stability constants and modelling of metal accumulation. Can. J. Fish. Aquat. Sci. 50,2678 -2687.
Reid, S. D. and McDonald, D. G. (1988). Effects of cadmium, copper, and low pH on ion fluxes in the rainbow trout, Salmo gairdneri. Can. J. Fish. Aquat. Sci. 45,244 -253.
Richards, J. G. and Playle, R. C. (1999). Protective effects of calcium against the physiological effects of exposure to a combination of cadmium and copper in rainbow trout. Can. J. Fish. Aquat. Sci. 77,1035 -1047.
Rogers, J. T., Richards, J. G. and Wood, C. M. (2003). Ionoregulatory disruption as the acute toxic mechanism for lead in the rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 64,215 -234.[CrossRef][Medline]
Sayer, M. D. J., Reader, J. P. and Morris, R. (1989). The effect of calcium concentration on the toxicity of copper, lead and zinc to yolk-sac fry of brown trout (Salmo trutta L.) in soft, acid water. J. Fish. Biol. 35,323 -332.
Sayer, M. D. J., Reader, J. P. and Morris, R. (1991). Effects of six trace metals on calcium fluxes in brown trout (Salmo trutta L.) in soft water. J. Comp. Physiol. B 161,537 -542.[Medline]
Schecher, W. D. and McAvoy, D. C. (1994). MINEQL+: A Chemical Equilibrium Program for Personal Computers (Version 3.01). Hallowell, ME, USA: Environmental Research Software.
Segel, J. H. (1976). Biochemical Calculations: How to Solve Mathematical Problems in General Biochemistry, 2nd Edn. 496pp. New York: John Wiley & Sons Inc.
Settle, D. M. and Patterson, C. C. (1980). Lead in albacore: Guide to lead pollution in Americans. Science 207,1167 .[Medline]
Sorensen, E. M. B. (1991). Lead. In Metal Poisoning in Fish, pp.95 -118. Boca Raton: CRC Press Inc.
Spry, D. J. and Wood, C. M. (1985). Ion flux rates, acid-base status, and blood gases in rainbow trout, Salmo gairdneri, exposed to zinc in natural soft water. Can. J. Fish. Aquat. Sci. 42,1332 -1340.
Spry, D. J. and Wood, C. M. (1989). A kinetic method for the measurement of zinc influx in vivo in the rainbow trout, and the effects of waterborne calcium on flux rates. J. Exp. Biol. 142,425 -446.
US Environmental Protection Agency (1986). Air Quality Criteria For Lead. Research Triangle Park, NC: US Environmental Protection Agency, Office of Health and Environment Assessment, Environmental Criteria and Assessment Office. EPA 600,8-83-028F .
Varanasi, U. and Gmur, D. J. (1978). Influence of waterborne and dietary calcium on uptake and retention of lead by coho salmon (Oncorhynchus kisutch). Toxicol. Appl. Pharmacol. 46,65 -75.[Medline]
Verbost, P. M., Bryson, S. E., Wendelaar-Bonga, S. E. and Marshall, W. S. (1997). Na+-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus. J. Comp. Physiol. B 167,205 -212.[Medline]
Verbost, P. M., Flik, G., Fenwick, J. C., Greco, A. M., Pang, P. K. T. and Wendelaar-Bonga, S. E. (1993). Branchial calcium uptake: possible mechanism of control by stanniocalcin. Fish Physiol. Biochem. 11,205 -215.
Verbost, P. M., Flik, G., Lock, R. A. C. and Wendelaar-Bonga, S. E. (1987). Cadmium inhibition of Ca2+ uptake in rainbow trout gills. Am. J. Physiol. 253,R216 -R221.[Medline]
Verbost, P. M., Van Rooij, J., Flik, G., Pang, P. K., Lock, R. A. C. and Wendelaar-Bonga, S. E. (1989). The movement of cadmium through freshwater trout branchial epithelium and its interference with calcium transport. J. Exp. Biol. 145,185 -197.
Verbost, P. M., Schoenmakers, T. J. M., Flik, G. and
Wendelaar-Bonga, S. E. (1994). Kinetics of ATP- and
Na+-gradient driven Ca2+ transport in basolateral
membranes from gills of freshwater- and seawater-adapted tilapia.
J. Exp. Biol. 186,95
-108.
Weiss, G. B. (1974). Cellular pharmacology of lanthanum. Annu. Rev. Pharmacol. 14,343 -354.
World Health Organization (1995). Environmental Health Criteria 165. Geneva: International Programme on Chemical Safety.
Wood, C. M., Hogstrand, C., Galvez, F. and Munger, R. S. (1996). The physiology of waterborne silver toxicity in freshwater rainbow trout (Oncorhynchus mykiss) 1. The effects of ionic Ag+. Aquat. Toxicol. 35,111 -125.[CrossRef]
Zohouri, M. A., Pyle, G. G. and Wood, C. M. (2001). Dietary Ca inhibits waterborne Cd uptake in Cd-exposed rainbow trout, Oncorhynchus mykiss. Comp. Biochem. Physiol. 130C,347 -356.