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First published online September 14, 2007
Journal of Experimental Biology 210, 3387-3394 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008748
Nitric oxide formation from nitrite in zebrafish
Institute of Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
e-mail: fbj{at}biology.sdu.dk
Accepted 18 July 2007
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Summary |
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Key words: hemoglobin, methemoglobin, nitric oxide, nitrite, nitrosylhemoglobin, oxygen consumption, spectral deconvolution
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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).
Nitrite is, however, also a natural constituent in the body, and recent
research has suggested that nitrite has important biological functions at its
natural low endogenous concentrations
(Gladwin et al., 2005). Thus,
nitrite may participate in local blood flow regulation (hypoxic vasodilation),
protect against ischemic insults, and it may act as a signaling molecule and
regulate gene expression (Bryan et al.,
2005; Gladwin et al.,
2005). Nitrite physiology therefore seems to be a balance between
toxic disruption of functions at high concentrations and beneficial effects at
low concentrations (Jensen,
2007).
Nitrite is produced inside the body as an oxidative metabolite of the
physiological messenger molecule nitric oxide (NO). Nitric oxide produced in
the vascular endothelium exerts its biological function by diffusing to the
underlying vascular smooth muscle, causing their relaxation and thus local
vasodilation. NO diffusing into the blood can react with plasma oxygen and
form nitrite. Other sources of nitrite include intake via the diet
and (in mammals) reduction of nitrate (present in diet or drinking water) to
nitrite by bacteria in the oral cavity
(Lundberg and Weitzberg,
2005). An important additional route in fish is the direct uptake
of nitrite from the ambient water to the blood across the gills. Nitrite has
an affinity for the active branchial chloride uptake mechanism in freshwater
fish, whereby even minor elevations of nitrite in the environment can lead to
massive accumulation of nitrite in the fish
(Margiocco et al., 1983;
Jensen, 2003).
Nitrite originating from NO oxidation was considered relatively inert,
until it became clear that nitrite may function as a storage pool of NO
activity (Cosby et al., 2003).
Thus, NO can be regenerated from nitrite by non-enzymatic acidic reduction
(Zweier et al., 1999) and by
enzymatic reduction via xanthine oxidoreductase
(Millar et al., 1998) or
deoxygenated hemoglobin (Cosby et al.,
2003). These reactions are favored by low pH and low oxygen
tension (PO2), pointing to their possible role
in vasodilation during hypoxia and exhausting exercise
(Gladwin et al., 2005;
Gladwin et al., 2006;
Fago and Jensen, 2007). The
formation of NO from nitrite and its physiological role in blood flow
regulation has mainly been examined and documented in mammalian models
(Cosby et al., 2003;
Crawford et al., 2006), but
some information is also available for fish
(Aggergaard and Jensen, 2001;
Jensen and Agnisola,
2005).
NO formation from nitrite is favored not only by low pH and
PO2 but also by high
[NO2–]. This is seldom considered, because nitrite
typically is present in the sub-micromolar or low micromolar range in plasma
or tissues (Kleinbongard et al.,
2003). However, in nitrite-exposed fish, plasma
[NO2–] can increase to the millimolar range, and
it seems likely that this could lead to excess NO production
(Jensen, 2003). This implies
that disturbance of NO homeostasis may be a major impact of nitrite exposure
in fish. The main aim of the present study was to test the hypothesis that a
massive production of NO occurs in nitrite-exposed fish.
NO reacts with deoxygenated heme groups of hemoglobin (Hb) to form
iron-nitrosyl Hb (HbNO). The reaction is rapid and has a strong binding
constant, whereas the rate of dissociation is low
(Antonini and Brunori, 1971).
Thus, the level of HbNO can be used as a `meter' for the internal NO level
(Gladwin et al., 2005). The
present study therefore used the blood HbNO concentration as a biomarker for
the internal NO production and NO load. By performing spectral deconvolution
on spectra of hemolysates obtained from zebrafish exposed to three nitrite
levels for variable time periods, it was possible to evaluate dose- and
time-dependent changes in HbNO, methemoglobin (metHb), functional Hb and total
Hb during nitrite exposure.
It is well documented that blood gas transport is disturbed by increased
metHb levels and decreased total [Hb] during nitrite exposure
(Jensen, 2003), but little is
known about how this affects O2 consumption in fish. Metabolism may
also be influenced by inhibition of mitochondrial respiration via NO
formed from nitrite (Crawford et al.,
2006; Shiva et al.,
2007). Consequently, a further aim of the present study was to
evaluate whether overall metabolic rate, as reflected by routine oxygen
consumption, is influenced by nitrite exposure. Nitrite exposure can induce
K+ efflux from intracellular to extracellular compartments, which
has been hypothesized to cause an overall potassium deficit
(Knudsen and Jensen, 1997). A
final aim was, therefore, to analyze whole body ion content during nitrite
exposure. Zebrafish were chosen as experimental animals, because their high
activity level should make changes in metabolic rate from limitations in
O2 transport easy to detect. Furthermore, zebrafish are used as
model animals in many biological disciplines, and their rearing and
maintenance can be associated with occasional nitrite exposure, which (as in
nature) results from nitrite built-up during imbalance in bacterial
nitrification or denitrification processes
(Jensen, 2003). Thus, the
information on nitrite effects and tolerance in zebrafish appeared a relevant
side reward of using this species to study NO formation from nitrite in
fish.
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Materials and methods |
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Experimental groups and sampling of blood and whole animals
The experimental design involved three exposure groups. Zebrafish were
exposed to either (i) control water (no nitrite addition, measured
[NO2–] 
0.005 mmol l–1), (ii)
0.6 mmol l–1 nitrite or (iii) 2.0 mmol l–1
nitrite. The required amount of nitrite was added as dissolved
NaNO2, and the water nitrite concentration was confirmed by
spectrophotometric measurements (Gries reaction). The fish were sampled after
0, 1 and 2 days of exposure at each of these three nitrite conditions. Fish
exposed to 0.6 mmol l–1 nitrite were additionally sampled
after 5 days of exposure.
Upon sampling, individual fish were netted and anesthetized in MS-222 (ethyl 3-aminobenzoate methanesulfonate; Sigma, Steinheim, Germany). Following full anesthesia, the tail was cut posterior to the anal fin and blood was sampled into a heparinized capillary tube held toward the exposed caudal vessels. This resulted in the collection of 2–8 µl blood. Blood was immediately processed for spectral analysis (cf. below), and, at the same time, the whole fish (including the tail) was transferred to a mortar for instant freezing in liquid nitrogen and pulverization with a pestle. The powdered fish was transferred to a pre-weighed vial for determination of tissue wet mass. To each vial, 2 ml of 8% perchloric acid was added and samples were allowed to digest for 48 h with intermittent mixing for the extraction of ions. The homogenates were centrifuged and the supernatant was used for measurements of whole body ion concentrations. Potassium was measured by atomic absorption spectroscopy (AAnalyst 100, Perkin-Elmer, Waltham, MA, USA), sodium was measured using a flame photometer (Instrumentation Laboratory 243) and chloride was measured with a Radiometer (Copenhagen, Denmark) CMT 10 chloride titrator. Ion concentrations were expressed as mmol kg–1 wet mass.
Spectral scans and spectral deconvolution
Directly after blood sampling, an accurate amount of blood was pipetted
into 1 ml 20 mmol l–1 phosphate buffer (pH 7.3). Cell debris
was removed from the hemolysate by centrifugation (1 min at 12 750
g), and the supernatant was transferred to a 1 cm, 1 ml
cuvette. A spectral scan was made from 480 to 700 nm in 0.2 nm steps, using a
Cecil CE2041spectrophotometer (Cambridge, UK). Scans were completed in less
than 3 min from blood sampling to exclude any potential post-sampling changes
in concentration of methemoglobin or HbNO.
The hemoglobin (Hb) solution from each individual zebrafish was assumed to
be a mixture of oxygenated Hb (oxyHb), methemoglobin (metHb), iron-nitrosyl Hb
(HbNO) and deoxygenated Hb (deoxyHb). Using the Lambert-Beer law, the measured
absorbance (A) at any wavelength (
) will be the sum of the
contribution of each individual Hb species, according to:
 | (1) |
signifies the millimolar extinction coefficient of
the given Hb species at wavelength , and l is thickness of
the absorbing layer (=1 cm). The use of this equation to evaluate the
concentration of each Hb derivative required information on values for
each Hb type at all concerned wavelengths.
A reference spectrum for oxyHb was obtained by hemolysing 8 µl freshly
drawn zebrafish blood in 1.0 ml air-equilibrated
(PO2=155 mmHg; 1 mmHg=133.3 Pa) 20 mmol
l–1 phosphate buffer (pH 7.3). Cell debris was removed by
centrifugation (1 min at 12 750 g), and 900 µl supernatant
was transferred to a 1 cm, 1 ml cuvette. A spectral scan was made from 480 to
700 nm in 0.2 nm steps. The resulting oxyHb spectrum had the two
characteristic peaks situated at 540.8 nm and 576.6 nm. The Hb concentration
(mmol heme l–1) in the cuvette was calculated from the
absorbance at these two peaks and the reported millimolar extinction
coefficients of 13.8 and 14.6 l mmol–1 cm–1
(Antonini and Brunori, 1971). A
reference spectrum for metHb was obtained by adding 0.00014 g
K3[Fe(CN)6] to the oxyHb solution and mixing with a
spatula. The reaction was completed within 30 min. DeoxyHb was obtained by
adding a few crystals of sodium dithionite
(Na2S2O4) to an oxyHb solution and mixing
with a spatula. Nitrosyl hemoglobin (HbNO) was obtained by adding 1.0 ml of
pure NO gas to deoxyHb in a closed, gas tight 1 ml cuvette. The millimolar
extinction coefficients for each Hb type at all the concerned wavelengths were
computed from the reference spectra as
A/(Cxl), where C
is total [Hb] and l is 1 cm. The obtained reference spectra (
versus ) for the four zebrafish Hb derivatives are shown in
Fig. 1A.
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oxyHb, metHb,
HbNO and deoxyHb the independent variables.
The output of the fitting procedure was the values of
CoxyHb, CmetHb,
CHbNO and CdeoxyHb that minimizes the
sum of squares of differences between the observed spectrum and the calculated
fit. The spectra were fitted with the constraints that the concentration of
each Hb derivative should be larger or equal to zero. At first, all parameters
to be fitted (i.e. CoxyHb, CmetHb,
CHbNO and CdeoxyHb) were allowed to
vary, but if one of the parameters converged towards zero after one to three
iterations (which was often the case for CdeoxyHb, as
expected from the high PO2 in the hemolysate),
then this variable was set to zero, and the iteration procedure was
continued. The determined values of CoxyHb, CmetHb, CHbNO and CdeoxyHb were used to calculate total [Hb] in the original blood sample (the sum of the four Hb species, taking into account the dilution factor). The percentages of oxyHb, metHb, HbNO and deoxyHb in the sample were calculated from the fraction each Hb species constituted of the total Hb. The calculated values for metHb and HbNO reflected their original in vivo values in the blood, as these derivatives are stable within the time frame here considered (3 min). The sum of oxyHb and deoxyHb gave the functional (potential O2 carrying) Hb of the sample.
Oxygen consumption
Fish were anaesthetized in MS-222, weighed and placed in closed chambers
each holding three or four fish. The water volume of each chamber (around 260
ml) was determined by weighing the water required to fill the chamber while
containing the three or four fish. The chambers were submerged in a 26°C
thermostatted bath and covered to avoid visual disturbance but yet allowing
light to enter during the light phase of the 24 h cycle. Water was pumped from
an experimental aquarium through the chambers, using peristaltic pumps (type
110, Ole Dich Instrumentmakers, Denmark) with a flow rate of 25 ml
min–1. The ingoing water tube exited at the bottom of the
chambers (to create mixing) and the outlet from the chamber was in the top.
The fish were allowed to acclimate to chamber condition for at least 24 h
before the start of measurements. Measurements of oxygen consumption were
started 24 h before nitrite exposure (–24 h). A 1-ml water sample was
drawn through a needle inserted into the outlet from each chamber and oxygen
tension (PO2) was measured with Radiometer
(Copenhagen, Denmark) E5046 electrodes in D616 thermostatted (26°C) cells,
with signals displayed on PHM 73 monitors and REC 80 recorders. The flow
through the chambers was then stopped for exactly 30 min. 5 s following
re-commencement of flow a new 1-ml sample was drawn for
PO2 measurement. Fish movements and the
re-establishment of flow ensured that samples were of truly mixed chamber
water. The rate of oxygen consumption
(
O2) was
calculated as
(Vwx
o2x
PO2)/(txMb),
where Vw is chamber water volume, o2 is
O2 solubility at 26°C [1.64 µmol l–1
mmHg–1 (Boutilier et al.,
1984)], t is elapsed time and Mb is
fish body mass. Measurements of oxygen consumption were done at –24,
–18 and –1 h in control water. Nitrite was then added to the
experimental aquarium and further measurements were done at 3, 23 and 47 h of
nitrite exposure. Two different series examined exposure to 0.6 mmol
l–1 nitrite (N=6 chambers) and 2 mmol
l–1 nitrite (N=4 chambers).
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; Huang et al.,
2005).
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Methemoglobin was low (0.2% of total Hb) and unchanged with time in control zebrafish (Fig. 3). Exposure to 0.6 mmol l–1 nitrite induced a small increase in the mean metHb level, but the variability was large, and the increase failed to be significant. In zebrafish exposed to 2 mmol l–1 nitrite the blood metHb level increased significantly with time and values at day 1 (41% of total Hb) and day 2 (59% of total Hb) were also significantly higher than in the other groups (Fig. 3).
The cause of the large variability in metHb values of nitrite-exposed fish was analyzed using a box chart of the pooled metHb data for each of the three groups. In zebrafish exposed to 0.6 mmol l–1 nitrite, the mean values at days 1, 2 and 5 were similar (Fig. 3), but it was clear from the box chart that the fish could be divided into responding and non-responding animals (Fig. 4). Thus, more than half the fish exposed to 0.6 mmol l–1 nitrite had metHb levels that did not differ from the low level observed in controls, whereas the remaining fish in this group had metHb levels elevated to a variable degree (Fig. 4). Thus, the data were not normal distributed and the median differed substantially from the mean (Fig. 4). In zebrafish exposed to 2 mmol l–1 nitrite all individuals showed elevated metHb levels, but to a variable degree (Fig. 4). This was partly due to the significant increase in metHb from day 1 to day 2, but individual variation in response magnitude was still present.
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The pattern of changes in nitrosylhemoglobin showed some resemblance with that for metHb. Thus, HbNO was low and constant in controls, rose to a somewhat higher but constant level in fish exposed to 0.6 mmol l–1 nitrite, and increased to very high levels in fish exposed to 2 mmol l–1 nitrite (Fig. 5). This increase in HbNO was significant both with time and when compared to the other groups at each individual day. The HbNO level reached a surprising high value of 12% of the total Hb in zebrafish exposed to 2 mmol l–1 nitrite for 2 days (Fig. 5).
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O2 values showed
no significant differences with either time or between fish exposed to 0.6
mmol l–1 and 2 mmol l–1 nitrite.
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In the blood, a main mechanism for HbNO formation will be the reaction of
nitrite with deoxygenated ferrous heme groups to form NO and ferric heme
(metHb), with NO subsequently being captured by unoxidized deoxygenated heme
groups to form nitrosylhemoglobin (Cosby et
al., 2003):
 | (2) |
 | (3) |
) and fish (F.B.J., unpublished data) Hbs. In addition to this
mechanism, some of the HbNO detected in the blood may result from reduction of
nitrite to NO via other heme proteins, xanthine oxidoreductase or
non-enzymatic acidic disproportionation
(Zweier et al., 1999;
Gladwin et al., 2005). The
latter will, however, require rather acidic conditions
(Modin et al., 2001), which
are not developed in nitrite-exposed cyprinids
(Jensen et al., 1987).
Whereas the reaction of nitrite with fully deoxygenated Hb leads to a 1:1
formation of HbNO and metHb (Eqn
2 and Eqn 3), the
in vivo situation is more complex. The blood O2 saturation
cycles between practically full saturation in arterial blood and intermediate
saturations in venous blood. At full O2 saturation, nitrite reacts
with oxyHb to form metHb and nitrate
(Kosaka and Tyuma, 1987),
whereas nitrite reacts with both oxyHb and deoxyHb at intermediate
O2 saturations (Grubina et al.,
2007) (F.B.J., unpublished). The production of HbNO is therefore
lower than that of metHb, even though HbNO formation stays significant even at
relatively high O2 saturations (F.B.J., unpublished). The oxidation
of Hb is countered in vivo by the presence of metHb reductase systems
inside the red blood cells, which can keep pace with the metHb formation when
nitrite concentrations are low. During nitrite exposure in fish, however, the
nitrite concentration continuously increases, and metHb levels are forced
upwards, albeit a quasi balance exist between Hb oxidation and Hb reduction at
each nitrite concentration (Jensen,
2003). On this background, the finding of higher metHb levels
(Fig. 3) than HbNO levels
(Fig. 5) in nitrite-exposed
zebrafish is expected. The astonishing result is the very high HbNO levels
that actually develop in the circulating blood
(Fig. 5).
In mammals, the basal concentration of HbNO is in the sub-micromolar range
but has been reported to increase to values ranging from 1 to 40 µmol
l–1 after administration of small doses of nitrite
via infusion, inhalation or food
(Cosby et al., 2003;
Hunter et al., 2004;
Tsuchiya et al., 2005). The
HbNO level of 12% reached in nitrite-exposed zebrafish
(Fig. 5) corresponds to some
400 µmol l–1 at the prevailing total [Hb]
(Fig. 2). This high level is
the result of the timely extended exposure to nitrite at much higher
concentrations than in mammals. Thus, whereas nitrite administration in
mammals typically leads to a transient increase in plasma nitrite in the
micromolar range, nitrite exposure in fish leads to a continuous increase in
plasma nitrite to values that can reach several millimolar
(Jensen, 2003).
The HbNO level in control zebrafish was about 1% of total Hb
(Fig. 5). This may, however,
not be the real basal level. Some of the control fish had HbNO levels
indistinguishable from zero, whereas HbNO was detectable in others, producing
the average of 1%. Control animals were kept in aquaria without biological
filters, and the measured water nitrite concentration was 1–5 µmol
l–1 in spite of the daily changes of water. Because zebrafish
could be divided into responding and non-responding animals (cf. results and
below), it seems plausible that responding fish took up some nitrite from the
low concentration in the control water, which would slightly elevate internal
nitrite and HbNO without affecting control metHb (due to red blood cell metHb
reductase activity). That NO formation may precede significant elevations of
metHb is supported by in vivo observations. In rainbow trout, heart
rate increases early during nitrite exposure, before metHb elevation or other
physiological disturbances are significant
(Aggergaard and Jensen, 2001).
It was suggested that the appearance of small amounts of nitrite in the blood
causes NO formation that triggers vasodilation and a decrease in blood
pressure, which quickly becomes countered by increased cardiac pumping to
re-establish blood pressure (Aggergaard and
Jensen, 2001).
A major fraction of the NO formed during nitrite exposure is either
captured by ferrous deoxyHb, forming HbNO, or reacts with oxyHb to produce
metHb and nitrate. These NO scavenging reactions of Hb limit the amount of
free NO (Kim-Shapiro et al.,
2006). Furthermore, some NO reacts with thiol groups to form
nitrosothiols (RSNO). NO can subsequently be released from HbNO and RSNO
compounds to some extent (Grubina et al.,
2007; Kim-Shapiro et al.,
2006). Indeed, the deoxyHb-mediated formation of NO is believed to
participate in local blood flow regulation through release of some of the NO
from the red cells (Cosby et al.,
2003). The very high HbNO level in nitrite-exposed zebrafish
testifies to a substantial production of NO from nitrite, creating a NO load
that probably disturbs normal NO homeostasis. Thus, the multitude of functions
that are influenced by NO, ranging from blood flow regulation and hemostasis
to neurotransmission, may all potentially be affected by excess NO production.
At present, however, little is known about these consequences. The rapid rise
in heart rate in rainbow trout lends some support to an interference of
nitrite-derived NO with cardiovascular function
(Aggergaard and Jensen, 2001).
NO inhibits platelet aggregation in mammals, and with the similarities of
hemostatic pathways and thrombocyte function in mammals and fish
(Jagadeeswaran et al., 1999),
one may hypothesize that excess NO formation during nitrite exposure could
increase bleeding from damaged blood vessels. Blood [Hb] decreases during
nitrite exposure (Fig. 2),
which has been proposed to reflect an increased removal of damaged red blood
cells from the circulation (Jensen,
2003). It may be speculated that internal bleeding could also
contribute. Evaluation of this and other potential disturbances of NO
homeostasis must, however, await further study.
Blood gas transport and oxygen consumption
The formation of high metHb levels (Fig.
3), with a consequent reduction in arterial oxygen content, are
well-known effects in nitrite-exposed fish (cf.
Jensen, 2003). A lowering of
total Hb, as observed in zebrafish exposed to 2 mmol l–1
nitrite (Fig. 2), has also been
reported in other fish, and will contribute to limitations in blood
O2 transport (Jensen,
2003). An important new realization from the present study is that
nitrosyl hemoglobin also contributes significantly to reduce blood
O2 capacitance. Owing to the tight binding and low rate of
dissociation of NO from HbNO, this Hb derivative has a long half-life and does
not participate in O2 transport. The high levels of HbNO
(Fig. 5) therefore add to the
high metHb levels (Fig. 3) in
lowering the amount of functional Hb (Fig.
6) in nitrite-exposed fish. Earlier studies have only considered
metHb formation and have therefore underestimated the amount of non-functional
Hb.
On basis of the declines in functional Hb and total Hb in nitrite-exposed
zebrafish, one may envisage that whole animal O2 consumption could
be reduced. The routine oxygen uptake was, however, not influenced by nitrite
exposure (Fig. 7). Absolute
values varied around 15 µmol h–1 g–1,
which compares well with earlier reported
O2 values in
zebrafish (Lucas and Priede,
1992). Thus, it appears that neither the reduction of arterial
O2 content nor the possibility that the internal NO load could
inhibit mitochondrial respiration (Crawford
et al., 2006; Shiva et al.,
2007) were of sufficient magnitudes to lower routine O2
consumption. It is predictable, however, that exercise capability and hypoxia
tolerance will be compromised by the lowered amount of functional Hb.
Nitrite tolerance in zebrafish
Zebrafish appeared relatively robust towards nitrite exposure. The mean
metHb level did not exceed 10% of total Hb during exposure to 0.6 mmol
l–1 nitrite (Fig.
3). Many other fish species would develop much higher metHb levels
when exposed to 0.6 mmol l–1 nitrite in water of the present
ionic composition. When ambient nitrite was elevated to 2 mmol
l–1, the metHb content in zebrafish blood rose to 59% in 2
days (Fig. 3). For comparison,
metHb rose to 83% in carp after 2 days exposure to a lower nitrite
concentration (1 mmol l–1), even though water
[Cl–] was higher (which limits nitrite uptake)
(Jensen et al., 1987), and in
rainbow trout metHb also increased to higher values than in zebrafish at lower
ambient nitrite and higher ambient [Cl–]
(Aggergaard and Jensen, 2001).
Carp is normally considered rather resistant towards nitrite, whereas rainbow
trout is fairly sensitive. The above comparison therefore reveals a
comparatively large tolerance towards nitrite in zebrafish. This is supported
by toxicological data that show high LC50 values in zebrafish
(Voslárova et al.,
2006). The underlying mechanism could be a relatively low
Cl– uptake rate across the gills in zebrafish, because it is
known that the branchial Cl– uptake rate is lower in
resistant than in sensitive fish species
(Williams and Eddy, 1986;
Tomasso and Grosell, 2005).
Alternatively, the affinity of nitrite for the active branchial
Cl– uptake mechanism could be reduced in zebrafish.
Interestingly, zebrafish could be divided into responding and
non-responding individuals when exposed to 0.6 mmol l–1
nitrite (Fig. 4). In rainbow
trout, individuals can similarly be divided into two groups, with some
individuals showing faster nitrite accumulation and more pronounced
physiological disturbances than others
(Stormer et al., 1996;
Aggergaard and Jensen, 2001).
The intraspecific difference in trout correlated with higher branchial nitrite
uptake rates in the more sensitive individuals
(Jensen, 2003). A similar
explanation may be applicable to the individual difference observed in
zebrafish.
Nitrite exposure reduces the K+ content of skeletal muscle in
rainbow trout and carp (Stormer et al.,
1996; Knudsen and Jensen,
1997). The release of K+ to the extracellular space
elevates extracellular [K+]
(Jensen et al., 1987;
Stormer et al., 1996;
Knudsen and Jensen, 1997) but
less than expected from the amount of K+ released from skeletal
muscles, which suggests a further transport of K+ to the
environment and the development of a potassium deficit
(Knudsen and Jensen, 1997).
The whole body ion content was analyzed in zebrafish to test this possibility,
but a significant reduction in K+ content was not detected
(Fig. 8). It may be that the
high tolerance of zebrafish also correlates with a reduced K+
release compared to rainbow trout and carp.
Concluding remarks
By using the blood level of HbNO as a biomarker of NO production, the
present study documents an extensive in vivo formation of NO in
nitrite-exposed zebrafish. The main mechanism is the reduction of nitrite to
NO by deoxygenated heme groups inside red blood cells, with the subsequent
binding of NO to adjacent deoxygenated ferrous heme groups, forming HbNO. This
suggests that deoxyHb-mediated nitrite reduction is significant in the in
vivo arterial–venous circulation, where Hb cycles between full and
intermediate O2 saturations. This mechanism has been proposed to be
involved in blood flow regulation at natural low nitrite concentrations
(Cosby et al., 2003), but the
excess NO formation in nitrite-exposed zebrafish predicts that disturbance of
NO homeostasis is part of the toxic action of nitrite at high
concentrations.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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), 0.6 mmol l–1 nitrite (•) and 2 mmol
l–1 nitrite (
) for variable time periods. Values are
means ± s.e.m. (5
