Aquatic Toxicology 82 (2007) 204–213

Ammonia toxicity and tolerance in the brain of the

African sharptooth catfish, Clarias gariepinus
Nicklaus L.J. Wee a , Yvonne Y.M. Tng a , Hin T. Cheng a , Serene M.L. Lee a ,
Shit F. Chew b , Yuen K. Ip a,∗
a Department of Biological Sciences, National University of Singapore, Kent Ridge, Singapore 117543, Republic of Singapore
b Natural Sciences and Science Education, National Institute of Education, Nanyang Technological University,

1 Nanyang Walk, Singapore 637616, Republic of Singapore

Received 7 December 2006; received in revised form 9 February 2007; accepted 22 February 2007

Abstract
The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air-breather, and can survive on land during drought. The
objective of this study was to elucidate the mechanism of acute ammonia toxicity in C. gariepinus, and to examine whether methionine sulfoximine
[MSO; an inhibitor of glutamine synthetase (GS)] or MK801 [an antagonist of N-methyl d-aspartate type glutamate (NMDA) receptors] had
protective effects against acute ammonia toxicity in this fish. After 48 h of exposure to a sublethal concentration (75 mmol l−1 ) of environmental
ammonia, the brain glutamine and ammonia contents in C. gariepinus increased to 15 ␮mol g−1 and 4 ␮mol g−1 , respectively. Thus, C. gariepinus
detoxified ammonia to glutamine and could tolerate high levels of glutamine in its brain. After C. gariepinus was injected intraperitoneally with a
sublethal dose of ammonium acetate (CH3 COONH4 ; 8 ␮mol g−1 fish) followed with emersion, brain ammonia and glutamine contents increased
continuously during the subsequent 24-h period, reaching 7 and 18 ␮mol g−1 , respectively, at hour 24. These results suggest that when confronted
with acute ammonia toxicity, the survival of C. gariepinus was crucially determined by its high tolerance of ammonia and high capacity to detoxify
ammonia to glutamine in the brain. For fish injected with a sublethal dose of CH3 COONH4 (10 ␮mol g−1 fish) followed with immersion, there
were transient but significant increases in brain ammonia and glutamine contents, which peaked at hour 2 (4 ␮mol g−1 ) and hour 6 (6 ␮mol g−1 ),
respectively. From these results, it can be deduced that C. gariepinus accumulated glutamine in preference to ammonia in its brain. By contrast, for
fish injected with a lethal dose (20 ␮mol g−1 fish) of CH3 COONH4 followed with immersion, the brain ammonia content increased drastically to
10 ␮mol g−1 after 30 min, while the brain glutamine content remained relatively low at 5 ␮mol g−1 . Therefore, it can be concluded that increased
synthesis and accumulation of glutamine in the brain was not the major cause of death in C. gariepinus confronted with acute ammonia toxicity. The
determining factor of acute ammonia toxicity appeared to be the rate of ammonia build-up in the brain. MK801 (2 ␮g g−1 fish) had no protective
effect on C. gariepinus injected with a lethal dose of CH3 COONH4 (20 ␮mol g−1 fish) indicating that activation of NMDA receptors might not
be involved. By contrast, the prior administration of MSO (100 ␮g g−1 fish) reduced the mortality rate from 100% to 80% and at the same time
prolonged the time of death significantly from 27 min to 48 min. However, the protective effect of MSO was apparently unrelated to the inhibition
of glutamine synthetase and prevention of glutamine accumulation in the brain. Instead, MSO affected activities of glutamate dehydrogenase and
alanine aminotransferase and suppressed the rate of ammonia build up in the brain of fish injected with a lethal dose of CH3 COONH4 .
© 2007 Elsevier B.V. All rights reserved.

Keywords: Ammonia; Ammonia toxicity; Brain; Clarias gariepinus; Glutamine; Glutamine synthetase; MSO; MK801; Nitrogen metabolism

1. Introduction Randall, 1979; McKenzie et al., 1993), hyper-excitability, coma,

Ammonia is toxic to fish because of many reasons. At the
organismal level, ammonia causes hyperventilation (Hillaby and
∗ Corresponding author. Tel.: +65 6516 2702; fax: +65 6779 2486.
E-mail address: dbsipyk@nus.edu.sg (Y.K. Ip).
convulsions and finally death. It also affects the ionic balance
in fish, because NH4 + can substitute for K+ in Na+ /K+ -ATPase
and in Na+ /K+ /2Cl− co-transporter (see Wilkie, 1997, 2002 for
reviews; Person Le Ruyet et al., 1997). NH4 + can substitute
for H+ in Na+ /H+ exchanger (NHE), probably NHE2 and/or
NHE3 (Randall et al., 1999). At the cellular level, ammonia can
interfere with energy metabolism through impairment of the tri-

0166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2007.02.015
 N.L.J. Wee et al. / Aquatic Toxicology 82 (2007) 204–213
carboxylic acid cycle in fish (Arillo et al., 1981). In addition,
NH4 + can substitute for K+ (Binstock and Lecar, 1969) affecting
the membrane potential. Smart (1978) suggested that the mech-
anism of ammonia toxicity in fish might be similar to that in
mammals during hepatic encephalopathy. Several theories, i.e.
glutamatergic dysfunction, glutamine accumulation leading to
astrocyte swelling, and/or activation of N-methyl-d-aspartate-
type glutamate (NMDA) receptors, have been proposed as
mechanisms involved in chronic and/or acute ammonia toxicity
in mammalian brains (Felipo et al., 1994; Margulies et al., 1999;
Hermenegildo et al., 2000; Desjardins et al., 2001; Brusilow,
2002; Felipo and Butterworth, 2002; Rose, 2002). However, it
has been proposed recently that mechanisms of and defence
against ammonia toxicity in brains of fishes with high ammonia
tolerance could be different from those in mammals (see Ip et
al., 2004a,b; Chew et al., 2006 for reviews).
In mammals, ammonia toxicity involves the activation
of NMDA receptors (Fan and Szerb, 1993; Hermenegildo
et al., 2000), and the administration of a NMDA receptor
antagonist, (5R, 10S)-(+)-methyl-10, 11-dihydro-5H-dibenzo[a,
d]cyclohepten-5,10-imine hydrogen maleate (MK801), at a
dosage of 2 ␮g g−1 animal delays or even eliminates the fatal
effects of ammonia toxicity in rats (Warren and Schenker, 1964;
Takahashi et al., 1991; Willard-Mack et al., 1996; Brusilow,
2002; Marcaida et al., 1992; Hermenegildo et al., 1996). While
a similar dosage of MK801 appears to have a protective effect
on the weatherloach (Tsui et al., 2004), such an effect cannot
be detected in two mudskippers (Periophthalmodon schlosseri
and Boleophthalmus boddarti) confronted with acute ammo-
nia toxicity (Ip et al., 2005b). Furthermore, ammonia toxicity
in mammals could be a result of increased glutamine pro-
duction and accumulation, which cause increased astrocyte
swelling, leading to cellular dysfunction, brain edema and
death (Brusilow, 2002). Indeed, l-methionine S-sulfoximine
(MSO) inhibits glutamine synthetase (GS), reduces edema,
attenuates ammonia-induced increases in brain extracellular
K+ , and ameliorates ammonia toxicity when administered at a
dosage of 100 ␮g g−1 to patients suffering from acute hepatic
encephalopathy (Brusilow, 2002). However, increases in glu-
tamine synthesis and accumulation occur in the brain of the Gulf
toadfish, Opsanus beta, without causing brain edema (Veauvy
et al., 2005); and, MSO does not have any significant protective
effects on mudskippers confronted with acute ammonia toxicity
(Ip et al., 2005b).
Many freshwater catfishes (members of families Clariidae
and Heteropneustidae) in the tropics are obligatory air-breathers;
they can survive on land for many days and exhibit high tolerance
of environmental ammonia. The African sharptooth catfish Clar-
ias gariepinus (Burchell) has, in addition to degenerate gills,
accessory breathing organs for air-breathing (Graham, 1997). It
is ammonotelic in water as urea-N accounts for only ∼10% of the
total waste-N (Eddy et al., 1980; Ip et al., 2004c, 2005a). Despite
being able to tolerate high concentrations of environmental
ammonia (Eddy et al., 1980; Ip et al., 2004c), C. gariepinus
does not detoxify ammonia to urea or amino acids (including
glutamine) during ammonia exposure (Ip et al., 2004c). It does
not possess a functional ornithine urea cycle, and no carbamoyl
205
phosphate synthetase III activity can be detected from its liver,
not even after exposure to 100 mmol l−1 NH4 Cl at pH 7.0 for 5
days (Ip et al., 2004c). Ip et al. (2004c) demonstrated that the
ability of C. gariepinus to survive in high concentrations of envi-
ronmental ammonia was partially related to its ability to actively
excrete ammonia, presumably NH4 + . Furthermore, Ip et al.
(2005a) reported that four days of emersion did not induce ureo-
genesis or ureotely in this catfish, and ammonia was not excreted
as NH3 through volatilization during emersion. In addition,
there were no changes in levels of alanine in the muscle, liver,
and plasma; neither were there any changes in the glutamine
levels in these tissues. More importantly, there were extraordi-
narily high levels of ammonia in the muscle (14 ␮mol g−1 ), liver
(18 ␮mol g−1 ) and brain (11 ␮mol g−1 ) of fish exposed to terres-
trial conditions for 4 days. Thus, Ip et al. (2005a) concluded that
C. gariepinus could tolerate high levels of ammonia at the cel-
lular and tissue levels, especially in the brain, during emersion.
That being the case, the mechanisms of ammonia toxicity in the
brain of C. gariepinus remains enigmatic because encephalopa-
thy would develop in mammalian brains with 1–3 ␮mol g−1
of ammonia (Cooper and Plum, 1987). Therefore, the present
study was undertaken to elucidate the mechanisms of ammonia
toxicity and tolerance in the brain of C. gariepinus.
Five sets of experiments were performed in this study. First,
experiments were undertaken to establish the levels of ammonia,
glutamine, glutamate, and alanine in the brains of fish immersed,
but with free access to air, in freshwater or water containing
sublethal concentrations (15 or 75 mmol l−1 ) of environmen-
tal ammonia for 48 h. The objectives of this set of experiments
were to determine how high the brain ammonia and glutamine
levels could be and to examine whether the brain glutamate
and alanine contents would decrease as a result of increased
glutamine synthesis during chronic exposure to environmental
ammonia. Second, experiments were undertaken to determine
contents of ammonia, glutamine, glutamate, and alanine in the
brains collected at various time points from fish that had been
injected intraperitoneally with a sublethal dose (8 ␮mol g−1
fish) of ammonium acetate (CH3 COONH4 ) followed by 24 h of
emersion. During emersion, ammonia could not be effectively
excreted and consequently the experimental fish had to defend
against the toxicity of almost all of the exogenous ammonia
injected into them. The objective of this set of experiments was
to evaluate the upper limits of ammonia and glutamine con-
tents in the brain of this fish. Third, fish were euthanized at
specific time points during 24 h of immersion after being injected
intraperitoneally with a sublethal dose (10 ␮mol g−1 fish) of
CH3 COONH4 . In addition to examining contents of ammonia
and glutamine in the brains of these fish, we aimed to determine
whether C. gariepinus, despite having degenerate gills, could up-
regulate ammonia excretion in water effectively to ameliorate the
toxicity of ammonia. Fourth, fish were euthanized for tissue sam-
pling 30 min after the intraperitoneal injection with a lethal dose
(20 ␮mol g−1 fish) of CH3 COONH4 followed by immersion. It
was hoped that results obtained from this set of experiments
would shed light on whether glutamine and/or ammonia accu-
mulation could be the cause of death in C. gariepinus confronted
with acute ammonia toxicity. Fifth, experiments were performed
206 N.L.J. Wee et al. / Aquatic Toxicology 82 (2007) 204–213
to evaluate whether MSO or MK801 had protective effects
on C. gariepinus injected intraperitoneally with a lethal dose
(20 ␮mol g−1 fish) of CH3 COONH4 followed by immersion.
2. Materials and methods
2.1. Experimental animals
Clarias gariepinus (80–200 g body mass) were purchased
from the China Town wet market, Singapore. They were main-
tained in plastic aquaria filled with dechlorinated tap water at
25 ◦ C in the laboratory, and water was changed daily. No attempt
was made to separate the sexes. The fish were acclimated to lab-
oratory conditions for at least three weeks. During the adaptation
period, C. gariepinus was fed an artificial diet. Food was with-
drawn 48 h prior to experiments, which gave sufficient time for
the gut to be emptied of all food and waste.
2.2. Experimental conditions
2.2.1. Immersion of fish in water containing 15 or
75 mmol l−1 NH4 Cl for 48 h
Experimental fish were immersed individually in 2 l of
dechlorinated water containing a sublethal dose (15 or
75 mmol l−1 ) of NH4 Cl in plastic tanks (L 28 cm × W 19 cm × H
18 cm) for 48 h, because Ip et al. (2004) reported that C. gariepi-
nus could survive in 100 mmol l−1 of NH4 Cl. There was no
need to aerate the water because C. gariepinus is an air-breather.
While immersed in water, fish had free access to the water sur-
face to breathe air. Control fish were immersed in dechlorinated
water for the same period. At the end of 48 h, fish were eutha-
nized and the brains were excised and freeze-clamped in liquid
nitrogen. Brain samples were kept at −80 ◦ C until the analysis
of ammonia, glutamine, glutamate and alanine.
2.2.2. Determination of sublethal and lethal dose of
CH3 COONH4 injected into the peritoneal cavity
To determine the 24 h sublethal and lethal dose of
CH3 COONH4 to be used in this study, 3 groups of 10 fish
each (a total of 30 fish) were anaesthetized for approximately
10 min in 0.1% phenoxyethanol (Sigma Chemical Co., St. Louis,
MO, USA) and injected intraperitoneally (0.5 ml 100 g−1 fish)
with 10, 15 or 20 ␮mol g−1 fish of CH3 COONH4 . Prior to the
experiment, all equipment were sterilized to prevent infection.
Control fish (N = 10) were injected with CH3 COONa followed
by immersion. Fish were then immersed in 2.5 l of strongly aer-
ated freshwater, and they usually recovered and responded to
mechanical stimulation within 7–10 min. For control fish, no
mortality was recorded at hour 24. For experimental fish, mor-
tality and time to death were recorded during the subsequent 24-h
period. Fish was regarded as dead when there was no observable
respiratory activity and no reaction to mechanical stimulation.
The sublethal and lethal doses of CH3 COONH4 for immersed
fish were 10 and 20 ␮mol g−1 fish, respectively. A similar set of
experiment was performed with fish exposed to 24 h of emersion
after the injection with CH3 COONH4 . The sublethal dose was
determined to be 8 ␮mol CH3 COONH4 g−1 fish.
2.2.3. Intraperitoneal injection with a sublethal dose
(8 μmol g−1 fish) of CH3 COONH4 followed by 24 h of
emersion
Fish were anaesthetized and injected intraperitoneally with
10 ␮mol g−1 fish of CH3 COONH4 or CH3 COONa (control).
Fish were subjected to emersion in plastic tanks (L 28 cm × W
19 cm × H 18 cm) containing only 200 ml of freshwater (pH
7.0). Under such terrestrial conditions, only the ventral surface
of the fish was in contact with a thin film of water. After 0.5,
2, 6, 12 or 24 h, fish were euthanized and brains were collected
and kept at −80 ◦ C until further analysis.
2.2.4. Intraperitoneal injection with a sublethal dose
(10 μmol g−1 fish) of CH3 COONH4 followed by 24 h of
immersion
Fish were anaesthetized and injected intraperitoneally with
10 ␮mol g−1 fish of CH3 COONH4 or CH3 COONa (control).
Fish were immersed individually in 2.5 l of freshwater (pH.
7.0) with free access to air in plastic containers (L 28 cm × W
19 cm × H 18 cm). After 0.5, 2, 6, 12 or 24 h, fish were eutha-
nized and brains were collected and kept at −80 ◦ C until further
analysis.
2.2.5. Intraperitoneal injection with a lethal dose
(20 μmol g−1 fish) of CH3 COONH4 and euthanized after
30 min of immersion
Fish were anaesthetized and injected intraperitoneally with
20 ␮mol g−1 fish of CH3 COONH4 or CH3 COONa (control).
Fish were immersed individually in 2.5 l of freshwater, and euth-
anized after 30 min. Brain samples were collected and kept at
−80 ◦ C until further analysis.
2.3. Determination of ammonia, glutamine, glutamate and
alanine contents
The frozen brain samples were weighed and ground to a
powder in liquid nitrogen. Five volumes (w/v) of ice-cold 6%
trichloroacetic acid (TCA) were added and the mixture was
homogenized three times for 20 s each (with 10 s off intervals)
with an Ultra-Turrax homogenizer at 24,000 revs min−1 . The
samples were then centrifuged for 20 min at 10,000 × g at 4 ◦ C
to obtain the supernatant.
For ammonia analysis, the pH of the supernatant was adjusted
to between 6.0 and 6.5 with 2 mol l−1 KHCO3 . The ammo-
nia content was determined using the enzymatic method of
Bergmeyer and Beutler (1985). The change in absorbance at
25 ◦ C and 340 nm was monitored using a Shimadzu UV-160A
spectrophotometer. Freshly prepared NH4 Cl solution was used
as the standard for comparison.
For glutamine, glutamate and alanine analyses, the super-
natant was diluted with an equal volume of 0.2 mol l−1
lithium citrate buffer and adjusted to pH 2.2 with 4 mol l−1
LiOH. The free amino acid contents in the tissues were
determined using a Shimadzu LC-6A Amino Acid Analy-
sis System (Kyoto, Japan) with a Shim-pack ISC-07/S1504
Li-type column. Despite complete FAA analyses being per-
formed on every sample, only contents (␮mol g−1 brain tissue)
 N.L.J. Wee et al. / Aquatic Toxicology 82 (2007) 204–213
of glutamine, glutamate and alanine were presented in this
report.
2.4. To evaluate the possible protective effects of MK801
and MSO on acute ammonia toxicity
2.4.1. Protective effect of MK801
The possible protective effect of MK801 (Sigma–Aldrich
Chemical Co., St. Louis, MI, USA) was evaluated by inject-
ing 2 ␮g g−1 fish of MK80l into the peritoneal cavity of fish
(N = 10 each) 15 min prior to the injection with a lethal dose
of CH3 COONH4 . Preliminary results indicated that the injec-
tion of 2 ␮g MK801 g−1 fish alone had no observable effects on
C. gariepinus. Controls (N = 10 each) were injected with 0.9%
NaCl solution in place of MK801 prior to the injection with
CH3 COONH4 .
2.4.2. Protective effect of MSO
To evaluate if MSO had a protective effect against acute
ammonia toxicity on C. gariepinus, fish (N = 10) were injected
intraperitoneally with 100 ␮g g−1 of MSO (pH 7.0) 15 min
prior to the injection with a lethal dose of CH3 COONH4
(20 ␮mol g−1 fish). Preliminary results indicated that injection
of 100 ␮g MSO g−1 fish alone had no observable effects on
C. gariepinus. Fishes (N = 10 each) injected with 0.9% NaCl
solution in place of MSO served as controls for comparison.
The mortality rate and time of death for succumbed fish were
recorded during the subsequent 24 h.
2.5. Effects of MSO on brain ammonia, glutamine and
glutamate contents and plasma ammonia concentration
Fish (N = 5 each) were injected intraperitoneally with
100 ␮g g−1 of MSO or 0.9% NaCl (control) 15 min prior to
the injection with a lethal dose of CH3 COONH4 (20 ␮mol g−1
fish), followed with immersion in 2 l of freshwater. Fish were
killed 30 min later. Brains were excised and kept at −80 ◦ C until
analysis. Contents of ammonia, glutamine and glutamate in the
brain were determined as described above. Blood samples were
collected from the severed caudal peduncle into heparinized
Eppendorf tubes and centrifuged at 5,000 × g at 4 ◦ C for 5 min to
obtain the plasma. The plasma was deproteinized by adding one
volume (v/v) of ice-cold 6% TCA and centrifuged at 10,000 × g
at 4 ◦ C for 15 min. The resulting supernatant was kept at −20 ◦ C
until ammonia analysis following the method described above.
2.6. Effects of MSO injected into the peritoneal cavity on
activities of some enzymes from the brain samples
Fish (N = 5 each) were injected intraperitoneally with
100 ␮g g−1 fish of MSO or 0.9% NaCl 15 min prior to the injec-
tion with a lethal dose of 20 ␮mol g−1 fish CH3 COONH4 . Fish
were killed 30 min after immersion in 2.5 l of freshwater. The
brain was quickly excised, freeze-clamped in liquid nitrogen,
and stored at −80 ◦ C until analysis.
The frozen brain, liver and muscle samples were weighed
and homogenized, using an Ultra-Turrax homogenizer, three
207
times in 5 volumes (w/v) of ice-cold extraction buffer con-
taining 50 mmol l−1 imidazole-HCl (pH 7.0), 50 mmol l−1 NaF,
3 mmol l−1 EGTA, 3 mmol l−1 EDTA at 24,000 revs min−1 for
20 s each with a 10 s off interval. The homogenate was cen-
trifuged at 10,000 × g at 4 ◦ C for 20 min. The supernatant
obtained was passed through a 5 ml Econo-Pac 10DG desalt-
ing column (Bio-Rad Laboratories Inc., CA, USA) equilibrated
with the same buffer solution. The resulting eluent was used for
enzyme assays. Protein concentrations of the extract were mon-
itored before and after filtration to determine the dilution factor
involved. Protein was determined according to the method of
Bradford (1976).
Glutamine synthetase (GS, EC 6.3.1.2) was determined as
transferase activity by the method of Shankar and Anderson
(1985) with some modifications. The reaction mixture consisted
of 67 mmol l−1 imidazole (pH 7.0), 25 mmol l−1 hydroxyla-
mine, 30 mmol−1 arsenate, 100 mmol l−1 glutamine, 1 mmol l−1
ADP, 1.7 mmol l−1 MnCl2 and 0.1 ml sample in a final volume
of 0.8 ml. After incubation at 25 ◦ C for 10 min, the reaction was
terminated with 0.4 ml of ferric chloride reagent (0.37 mmol l−1
FeCl3 , 0.20 mmol l−1 TCA and 0.67 mmol l−1 HCl). The solu-
tion was centrifuged at 10,000 × g at 4 ◦ C for 5 min to remove
precipitated proteins. Reaction terminated at time 0 was used
as blank. GS activity was measured at 500 nm and expressed
as ␮mol ␥-glutamyl hydroxamate formed min−1 mg−1 protein.
Freshly prepared glutamic acid ␥-monohydroxamate solution
was used as a standard for comparison.
Glutamic dehydrogenase (GDH, E.C. 1.4.1.3) was assayed
in the amination direction according to Ip et al. (1993) with
some modifications. The assay was performed in a total vol-
ume of 1.5 ml containing 200 mmol l−1 imidazole (pH 7.8),
10 mmol l−1 ␣- ketoglutarate, 1 mmol l−1 ADP, 0.17 mmol l−1
NADH and 0.1 ml sample. The reaction was initiated by the
addition of 0.2 ml of ammonium acetate at a final concentra-
tion of 25 mmol l−1 . Change in absorbance was monitored at
340 nm. Enzyme activity was expressed as ␮mol NADH oxi-
dised min−1 mg−1 protein.
Alanine aminotransferase (ALT, E.C. 2.6.1.2) in the direction
of alanine degradation was determined following the method of
Peng et al. (1994) in a reaction mixture of 1.2 ml containing
50 mmol l−1 imidazole (pH 7.4), 10 mmol l−1 ␣-ketoglutarate,
0.025 mmol l−1 pyridoxal phosphate, 0.15 mmol l−1 NADH,
1 i.u. LDH and 0.05 ml of sample. The reaction was initiated by
the addition of 0.2 ml of 0.2 mol l−1 l-alanine and the change
in absorbance was monitored at 340 nm. Enzyme activity was
expressed as ␮mol NADH oxidised min−1 mg−1 protein.
2.7. Effects of MSO in vitro on activities of some enzymes
from the brain samples
Brain samples were collected from control fish immersed
in fresh water, and enzyme extracts were prepared as described
above. To determine the in vitro effects of MSO, activities of GS,
GDH, and ALT were assayed as described above with or without
(blank) 30 min pre-incubation of the enzyme extract with 0.1 ml
of 2 mmol l−1 MSO (pH 7.0) in the assay medium prior to the
initiation of reactions.
208 N.L.J. Wee et al. / Aquatic Toxicology 82 (2007) 204–213

2.8. Statistics Table 1

Contents of ammonia, glutamine, glutamate and alanine in the brain (␮mol g−1
tissue) of Clarias gariepinus immersed for 48 h in freshwater (control) or water
Results were presented as means ± standard errors of mean containing a sublethal concentration (15 mmol l−1 or 75 mmol l−1 ) of NH4 Cl
(S.E.M.). Student’s t-test and one-way analysis of variance fol-
lowed by Bonferroni’s multiple range test were used to evaluate Control 15 mmol l−1 NH4 Cl 75 mmol l−1 NH4 Cl
differences between means where applicable. Differences with Ammonia 1.72 ± 0.12a 1.70 ± 0.16a 4.41 ± 0.68b
P < 0.05 were regarded as statistically significant. Glutamine 1.79 ± 0.16a 5.46 ± 0.87b 15.4 ± 1.5c
Glutamate 4.21 ± 0.69 4.84 ± 0.37 5.41 ± 0.45
Alanine 0.35 ± 0.07 0.31 ± 0.04 0.40 ± 0.02
3. Results
Results represent means ± S.E.M. (N = 4). Means not sharing the same letter are
significantly different, P < 0.05.
3.1. Contents of ammonia, glutamine, glutamate and
alanine

3.1.1. Fish exposed to environmental ammonia (15 or
75 mmol l−1 NH4 Cl) for 48 h
There was no mortality in fish exposed to 15 or 75 mmol l−1
NH4 Cl for 48 h. The ammonia content in the brain of fish
exposed to 15 mmol l−1 NH4 Cl for 48 h remained unchanged
but that of fish exposed to 75 mmol l−1 NH4 Cl increased sig-
nificantly (2.6-fold) to 4.41 ± 0.68 ␮mol g−1 tissue (Table 1).
The brain glutamine content of fish exposed to 15 and
75 mmol l−1 NH4 Cl increased significantly to 5.46 ± 0.87 (3-
fold) and 15.4 ± 1.5 (9-fold) ␮mol g−1 tissue, respectively
(Table 1). Despite a significant increase in glutamine content,
there were no significant changes in contents of glutamate
and alanine in the brain of fish exposed to 15 or 75 mmol l−1
NH4 Cl.
3.1.2. Fish injected intraperitoneally with a sublethal dose
(8 μmol g−1 fish) of CH3 COONH4 followed by 24 h of
emersion
No mortality was observed in fish injected intraperitoneally
with 8 ␮mol g−1 fish of CH3 COONH4 followed by 24 h of
emersion. There were significant increases in ammonia and glu-
tamine contents in the brain throughout the 24-h period, reaching
7.07 ± 0.82 and 17.72 ± 1.35 ␮mol g−1 tissue, respectively, in
the brains of the experimental fish at hour 24 (Table 2). No
significant changes in brain glutamate and alanine levels were
observed throughout the 24-h period in the experimental fish as
compared with the control.
3.1.3. Fish injected intraperitoneally with a sublethal dose
(10 μmol g−1 fish) of CH3 COONH4 followed by 24 h of
immersion
The injection with 10 ␮mol g−1 fish of CH3 COONH4 fol-
lowed by 24 h of immersion resulted in no mortality among the
experimental fish. However, it led to significant increases in the
contents of ammonia (4-fold) and glutamine (3.3-fold) in the
brain within the first 2 h (Table 3). The brain glutamine content
continued to increase and peaked (6.18 ± 0.38 ␮mol g−1 tissue)
at hour 6, even though the brain ammonia content returned
to a level comparable to that of the control. By contrast, the
brain glutamate content decreased significantly at hour 2, but
returned to control level at hour 6. There was no significant
change in the alanine content in the brain throughout the 24-h
period.
The peak levels of ammonia and glutamine in the brain of fish
injected with 10 ␮mol g−1 fish of CH3 COONH4 followed by
24 h of immersion (Table 3) were significantly lower (P < 0.05)
than those of fish injected with 8 ␮mol g−1 fish of CH3 COONH4
followed by 24 h of emersion (Table 2).
3.1.4. Fish injected intraperitoneally with a lethal dose
(20 μmol g−1 fish) of CH3 COONH4 and euthanized after
30 min of immersion
For C. gariepinus injected with a lethal dose of CH3 COONH4
and euthanized after 30 min of immersion, the ammonia and
glutamine levels were 9.63 ± 0.30 and 5.36 ± 0.32 ␮mol g−1

Table 2
Contents of ammonia, glutamine, glutamate and alanine in the brain (␮mol g−1 brain) of Clarias gariepinus after the intraperitoneal injection with 8 ␮mol g−1 fish
of CH3 COONa (control) or a sublethal dose (8 ␮mol g−1 fish) of CH3 COONH4 followed by 0.5, 2, 6, 12 or 24 h of emersion
Condition 0.5 h 2h 6h 12 h 24 h

Ammonia Control 1.32 ± 0.16 1.81 ± 0.09 2.31 ± 0.23 2.16 ± 0.12 2.07 ± 0.31
CH3 COONH4 4.10 ± 0.54a,* 6.23 ± 0.52b,* 5.26 ± 0.23a,* 7.36 ± 0.33b,* 7.07 ± 0.82b,*
Glutamine Control 1.93 ± 0.23a 2.87 ± 0.51a 3.58 ± 0.79a 5.99 ± 0.54b 7.53 ± 1.85b
CH3 COONH4 1.79 ± 0.32a 4.23 ± 1.11a 13.60 ± 0.62b,* 16.53 ± 1.32c,* 17.72 ± 1.35c,*
Glutamate Control 5.48 ± 0.27 4.86 ± 0.73 5.67 ± 1.11 5.34 ± 0.58 6.00 ± 0.85
CH3 COONH4 4.48 ± 0.40a 5.04 ± 0.47a 6.53 ± 0.46b 6.58 ± 0.34b 6.47 ± 0.52b
Alanine Control 0.40 ± 0.03 0.53 ± 0.12 0.45 ± 0.05 0.55 ± 0.09 0.47 ± 0.14
CH3 COONH4 0.53 ± 0.07 0.52 ± 0.04 0.39 ± 0.10 0.65 ± 0.10 0.67 ± 0.12

Results represent means ± S.E.M. (N = 4). (*) Significantly different from the corresponding control value, P < 0.05. Means not sharing the same letter are significantly
different, P < 0.05.
 N.L.J. Wee et al. / Aquatic Toxicology 82 (2007) 204–213 209

Table 3
Contents of ammonia, glutamine, glutamate and alanine in the brain (␮mol g−1 brain) of Clarias gariepinus after the intraperitoneal injection with 10 ␮mol g−1 fish
of CH3 COONa (control) or a sublethal dose (10 ␮mol g−1 fish) of CH3 COONH4 followed by 0.5, 2, 6, 12 or 24 h of immersion
Condition 0.5 h 2h 6h 12 h 24 h

Ammonia Control 1.10 ± 0.17 1.00 ± 0.10 1.21 ± 0.31 1.16 ± 0.09 0.98 ± 0.08
CH3 COONH4 3.50 ± 0.50b,* 3.91 ± 0.38b,* 1.09 ± 0.04a 1.04 ± 0.09a 1.15 ± 0.08a
Glutamine Control 2.03 ± 0.24 1.66 ± 0.08 1.62 ± 0.14 1.85 ± 0.03 2.26 ± 0.26
CH3 COONH4 3.33 ± 0.27a 5.42 ± 0.13b,* 6.18 ± 0.38b,* 3.28 ± 0.22a,* 2.65 ± 0.18a
Glutamate Control 6.15 ± 0.11 6.10 ± 0.42 5.73 ± 0.23 6.34 ± 0.45 5.61 ± 0.18
CH3 COONH4 5.21 ± 0.36 4.42 ± 0.11* 5.53 ± 0.22 5.21 ± 0.24 4.80 ± 0.13
Alanine Control 0.30 ± 0.09 0.22 ± 0.03 0.18 ± 0.02 0.22 ± 0.04 0.21 ± 0.02
CH3 COONH4 0.32 ± 0.06 0.20 ± 0.02 0.13 ± 0.01 0.17 ± 0.02 0.16 ± 0.03

Results represent means ± S.E.M. (N = 4). (*) Significantly different from the corresponding control value, P < 0.05. Means not sharing the same letter are significantly
different, P < 0.05.

Table 4
Contents of ammonia, glutamate and glutamine in the brain (␮mol g−1 tissue) and the concentration of ammonia in the plasma (␮mol ml−1 ) of Clarias gariepinus
30 min after the intraperitoneal injection with a lethal dose of 20 ␮mol CH3 COONH4 g−1 fish with the prior injection (15 min) of saline solution (0.9% NaCl; control)
or methionine sulfoximine (MSO; 100 ␮g g−1 fish)
Saline Saline + CH3 COONH4 (20 ␮mol g−1 fish) MSO + CH3 COONH4 (20 ␮mol g−1 fish)

Brain
Ammonia 1.04 ± 0.28a 9.63 ± 0.3c 5.95 ± 0.32b
Glutamine 2.29 ± 0.08a 5.36 ± 0.32b 6.16 ± 0.16b
Glutamate 6.49 ± 0.26a 6.44 ± 0.24a 7.39 ± 0.24b
Plasma
Ammonia 0.37 ± 0.02a 19.73 ± 1.65b 17.99 ± 0.78b

Results represent means ± S.E.M. (N = 4). Means not sharing the same letter are significantly different, P < 0.05.

tissue, respectively, which were significantly higher than
the corresponding values of the control (1.04 ± 0.28 and
2.29 ± 0.08 ␮mol g−1 tissue, respectively). Despite the sharp
increase in the brain ammonia level, the glutamate level in the
brain remained unaltered (Table 4).The brain ammonia con-
tent of the experimental fish injected with a lethal dose of
CH3 COONH4 followed by immersion (Table 4) was higher than
the peak levels of ammonia in the brain of fish injected with a
sublethal dose (8 ␮mol g−1 fish) of CH3 COONH4 followed by
24 h of emersion (Table 2) and fish injected with 10 ␮mol g−1
fish of CH3 COONH4 followed by 24 h of immersion (Table 3).
However, an opposite phenomenon was observed for the brain
glutamine content.
3.3.2. Effects of MSO on brain ammonia, glutamine and
glutamate contents and plasma ammonia concentration
The ammonia content in the brain of fish injected
with MSO followed by a lethal dose of CH3 COONH4
(5.95 ± 0.32 ␮mol g−1 tissue) was significantly lower than that
(9.63 ± 0.30 ␮mol g−1 tissue) of fish injected with a lethal dose
of CH3 COONH4 only (Table 4). However, the concentrations
of ammonia in the plasma of these two groups of fish were com-
parable (Table 4). Surprisingly, MSO had no significant effect
on the brain glutamine content of fish injected with a lethal dose
of CH3 COONH4 . However, there was a slight but significant
increase in the glutamate content in the brain of fish injected
with MSO and a lethal dose of CH3 COONH4 as compared with

3.2. Protective effect of MK801

Table 5
MK801 (2 ␮g g−1
fish) did not exert any protective effect Mortality (%) and time to death (min) of Clarias gariepinus injected intraperi-
toneally with a lethal dose (20 ␮mol g−1 fish) of CH3 COONH4 with the prior
on C. gariepinus injected with a lethal dose of CH3 COONH4 injection of saline solution (0.9% NaCl; control), MK801 (2 ␮g g−1 fish), or
(20 ␮mol g−1 fish) (Table 5). methionine sulfoximine (MSO; 100 ␮g g−1 fish)
Conditions Mortality (%) Time to death
3.3. Protective effect of MSO (min)

Control (Saline + CH3 COONH4 ) 100 26.7 ± 3.3a

3.3.1. Effects of MSO on mortality and time to death MK801 + CH3 COONH4 100 27.0 ± 2.7a
MSO (100 ␮g g−1 fish) reduced the mortality of fish injected MSO + CH3 COONH4 80 47.8 ± 6.8b
with 20 ␮mol CH3 COONH4 g−1 fish from 100% to 80%. Results of time to death represent means ± S.E.M. (N = 10), except for prior
In addition, it delayed the time to death significantly from injection of MSO (N = 8). Means not sharing the same letter are significantly
26.7 ± 3.3 min to 47.8 ± 6.8 min (Table 5). different, P < 0.05.
210 N.L.J. Wee et al. / Aquatic Toxicology 82 (2007) 204–213

Table 6 synthesis of glutamine following glutamate formation might

Specific activities (␮mol min−1 mg−1 protein) of glutamine synthetase (GS), be the reason for the reduction of cerebral ATP as observed in
glutamate dehydrogenase (GDH) in the amination direction and alanine amino-
transferase (ALT) from the brain sample of Clarias gariepinus 30 min after
ammonia-exposed rainbow trout (Arillo et al., 1981). However,
the intraperitoneal injection with a lethal dose of 20 ␮mol CH3 COONH4 g−1 in recent years, glutamatergic dysfunction, astrocyte swelling
fish with the prior injection (15 min) of saline solution (0.9% NaCl; control) or and brain herniation, and activation of N-methyl-d-aspartate-
methionine sulfoximine (MSO; 100 ␮g g−1 fish) type glutamate receptors related to ammonium ion induced
Enzyme Control MSO (Intraperitoneal Percentage membrane depolarization have been proposed as mechanisms of
injection) change acute ammonia toxicity in mammalian brains (Brusilow, 2002;
GS 0.31 ± 0.01 0.24 ± 0.01* −21.5%
Felipo and Butterworth, 2002; Rose, 2002).
GDH 0.21 ± 0.01 0.18 ± 0.02 NA The increased synthesis of glutamine (Meijer et al., 1990)
ALT 0.0179 ± 0.0014 0.0177 ± 0.0012 NA and its accumulation leading to astrocyte swelling is known to
Results represent means ± S.E.M. (N = 5). (*) Significantly different from the
be one of the reasons behind hepatic encephalopathy in mam-
corresponding control value, P < 0.05. NA = not applicable. mals (Brusilow, 2002). Fish brains are also known to detoxify
ammonia to glutamine (Peng et al., 1998; Ip et al., 2004a,b;
Table 7 Chew et al., 2006). By exposing the Gulf toadfish, O. beta, to
15 NH Cl, Rodicio et al. (2003) demonstrated that the glutamine
Specific activities (␮mol min−1 mg−1 protein) of glutamine synthetase (GS), 4
glutamate dehydrogenase (GDH) in the amination direction and alanine amino- in the brain became 15 N-labelled. Indeed, after 48 h of expo-
transferase (ALT) from the brain sample of Clarias gariepinus determined with sure to a sublethal concentration (75 mmol l−1 ) of environmental
or without 30 min of pre-incubation with methionine sulfoximine (MSO) in vitro
ammonia, the brain glutamine content in C. gariepinus increased
Enzyme Control MSO (in vitro) Percentage inhibition (%) to an exceptionally high level of 15.4 ␮mol g−1 . In compari-
GS 0.299 ± 0.013 0.166 ± 0.014* −44.9 son, the increase in the brain ammonia content (4.4 ␮mol g−1 )
GDH 0.188 ± 0.012 0.087 ± 0.005* −52.8 was much less drastic. Thus, C. gariepinus could up-regulate
ALT 0.016 ± 0.001 0.010 ± 0.001* −37.3 glutamine synthesis and tolerate high levels of brain glu-
Results represent means ± S.E.M. (N = 5). (*) Significantly different from the tamine with no mortality when exposed to 75 mmol l−1 NH4 Cl.
corresponding control value, P < 0.05. Similarly, Ip et al. (2005b) demonstrated that brains of mud-
skippers, P. schlosseri and B. boddarti, accumulated high levels
of glutamine (14–18 ␮mol g−1 ) after exposure to a sub-lethal
that of fish injected with saline and a lethal dose of CH3 COONH4
concentration (100 and 8 mmol l−1 , respectively, at pH 7.0)
(Table 4).
of environmental ammonia. Taken together, these results indi-
cate that increased glutamine synthesis and accumulation are
3.3.3. Effects of MSO on activities of enzymes from the not the major mechanisms of ammonia toxicity in these fishes.
brain samples In addition, using magnetic resonance imaging, Veauvy et al.
The specific activity of GS from the brain of fish injected (2005) demonstrated a lack of association between increase in
with MSO followed by a lethal dose of CH3 COONH4 was sig- brain glutamine level and brain hydration status in O. beta,
nificantly lower than that from the brain of fish injected with and concluded that increased glutamine synthesis and accu-
a lethal dose of CH3 COONH4 only (Table 6). However, MSO mulation did not cause cerebral swelling in this species. Even
apparently had no significant effects in vivo on the specific activ- with swelling, the presence of excess cranial space in fishes
ities of GDH and ALT from the brain of fish injected with a lethal in general would prevent the build up of pressure therein (Ip
dose of CH3 COONH4 (Table 6). et al., 2005b). It is important to note that increased glutamine
synthesis in C. gariepinus exposed to environmental ammonia
3.3.4. In vitro effects of MSO on activities of enzymes from did not result in a decrease in glutamate or alanine content,
the brain samples which suggests that there must be an increase in the synthe-
The specific activities of GS, GDH and ALT from brain sis of glutamate. If indeed that was the case, C. gariepinus
samples of C. gariepinus were reduced significantly when deter- probably could resolve problems associated with redox balance
mined in the presence of MSO in vitro, with 44.9%, 52.8%, and and the depletion of ␣-ketoglutarate during the increased detox-
37.3% of inhibition, respectively (Table 7). ification of ammonia to glutamate in its brain. Furthermore,
the lack of increase in alanine content suggests that increased
4. Discussion glutamate synthesis proceeded in tandem with increased glu-
tamine synthesis and was separated from other transamination
4.1. The brain of C. gariepinus could tolerate high levels of reactions.
glutamine and ammonia when exposed to a sublethal
concentration of environmental ammonia 4.2. Increased glutamine synthesis and accumulation was
not the major cause of death in fish confronted with acute
It was previously thought that ammonia toxicity was due to ammonia toxicity
the disruption of redox and depletion of ␣-ketoglutarate in the
brain when ammonia was converted to glutamate to remove In addition to high tolerance of environmental ammonia, C.
ammonia. Furthermore, the increase in ATP demand for the gariepinus could tolerate high dose of CH3 COONH4 injected
 N.L.J. Wee et al. / Aquatic Toxicology 82 (2007) 204–213
into its peritoneal cavity followed by emersion or immersion.
With immersion, it had a 24 h LC50 value of 15 ␮mol g−1 fish
(N. L. J. Wee and Y.K. Ip, unpublished results), which is com-
parable to that (15.6 ␮mol g−1 fish) of the giant mudskipper,
P. schlosseri (Ip et al., 2005b). C. gariepinus could continue
to excrete excess ammonia injected into its peritoneal cavity
during subsequent immersion, but ammonia excretion would
become relatively ineffective during emersion (Ip et al., 2005a).
Indeed, with emersion, it had a 24 h LC50 value of 12 ␮mol g−1
fish (Y.K. Ip, unpublished results). After fish were injected
with a sublethal dose of CH3 COONH4 (8 ␮mol g−1 fish)
followed by emersion, the ammonia and glutamine contents
in the brain increased continuously during the subsequent
24-h period, reaching 7 and 18 ␮mol g−1 , respectively, at hour
24. By contrast, mammals would succumb to encephalopathy
at a brain ammonia content of 1–3 ␮mol g−1 (Cooper and
Plum, 1987; Felipo and Butterworth, 2002). These results
suggest that when confronted with acute ammonia toxicity,
the survival of C. gariepinus was crucially dependent on its
high tolerance of ammonia and high capacity to detoxify
ammonia to glutamine in the brain, and corroborate in essence
the proposition that increased glutamine synthesis and accu-
mulation are not the major mechanism of ammonia toxicity in
C. gariepinus.
After C. gariepinus was injected with a sublethal dose of
CH3 COONH4 (10 ␮mol g−1 fish) followed with immersion,
there was a transient but significant increase in ammonia level
in the brain between hours 0.5 and 2, reaching 3.9 ␮mol g−1 .
The transient nature of ammonia accumulation could be a result
of the continual excretion of ammonia during immersion. There
was also a significant increase in the brain glutamine content
between hours 2 and 12, with a peak of 6.2 ␮mol g−1 at hour 6.
From these results, it can be deduced that C. gariepinus accu-
mulated glutamine in preference to ammonia in its brain, and
the content of the latter was maintained lower than that of the
former whenever possible. At hour 2, there was a transient but
significant decrease in glutamate content in the brain, which indi-
rectly implies that the rate of glutamine synthesis momentarily
outpaced the rate of glutamate production.
By contrast, for fish injected with a lethal dose (20 ␮mol g−1
fish) of CH3 COONH4 followed with immersion, the brain
ammonia content increased drastically to 9.6 ␮mol g−1 after
30 min, while the brain glutamine content remained relatively
low at 5.4 ␮mol g−1 . Therefore, it can be concluded that
increased synthesis and accumulation of glutamine in the brain
was not the major cause of death in C. gariepinus confronted
with acute ammonia toxicity. The brain ammonia level of
9.6 ␮mol g−1 in fish injected with a lethal dose of ammonia was
actually lower than that (11 ␮mol g−1 ) reported for fish exposed
to 4 days of emersion (Ip et al., 2005a). However, it is impor-
tant to consider the fact that the ammonia level was reached
within a 30-min period in the former fish injected with a lethal
dose of ammonia. Taken together, it can be deduced that one of
the determining factors of acute ammonia toxicity was the rate
at which ammonia built up in the brain. There could be some
degree of plasticity because C. gariepinus was able to accom-
modate a higher level of ammonia in its brain when the increase
211
in ammonia level happened to be gradual (e.g. through a 4-day
period) as in the case of emersion.
In addition, it has been well established in mammals that
the normal brain ammonia level is approximately 2 times that
of the blood because of the pH gradient involved (Cooper and
Plum, 1987). In contrast, for C. gariepinus injected intraperi-
toneally with a lethal dose of CH3 COONH4 , the brain ammonia
content (9.6 ␮mol g−1 ) was only half of the plasma ammonia
concentration (19.7 ␮mol ml−1 ). How the brain of C. gariepinus
confronted with acute ammonia toxicity maintains its ammo-
nia content to be lower than that of the plasma awaits future
studies.
4.3. MK801 did not exhibit a protective effect against acute
ammonia toxicity
Mortality in mammals, rats in particular, confronted with
acute ammonia toxicity could be prevented by the administration
of a wide range of NMDA receptor antagonists (Marcaida et al.,
1992; Hermenegildo et al., 1996). Studies using in vivo cerebral
microdialysis show that acute ammonia exposure results in the
activation of NMDA receptor-coupled nitric oxide-cyclic GMP
signal transduction pathway in the rat brain (Hermenegildo et
al., 2000). An increase in intracellular NH4 + can also lead to a
depolarization of membrane potential (Sugden and Newsholme,
1975), and depolarization can result in the reversal of glutamate
transport resulting in an increase in the extracellular gluta-
mate concentration (Szatkowski et al., 1990). Besides increasing
extracellular glutamate concentration, changes in membrane
potential can lead to the removal of Mg2+ block on NMDA
receptors and hence its activation (Fan and Szerb, 1993). The
deleterious effects of ammonia toxicity in mouse could be
ameliorated through the administration of MK801, a NMDA
receptor antagonist. At a dosage of 2 ␮g g−1 , MK801 provides
significant protection in animals injected intraperitoneally with
a lethal dose of ammonia, and there is a significant improvement
in clinical grading with lower electroencephalogram activity
(Marcaida et al., 1992).
By contrast, MK801 (2 ␮g g−1 fish) had no protective effect
on C. gariepinus injected with a lethal dose of CH3 COONH4
(20 ␮mol g−1 fish). Because of the negative result, it became
essential to verify that the same batch of MK801 exhibited a pro-
tective effect on another animal species. Indeed, when the same
batch of MK801 was tested at the same dosage on the Chinese
soft-shelled turtle, Pelodiscus sinensis, there was a significant
delay in the time to death for turtle injected intraperitoneally
with a lethal dose of CH3 COONH4 (S. M. L. Lee and Y. K. Ip,
unpublished results). In view of the relatively high brain ammo-
nia content in C. gariepinus confronted with acute ammonia
toxicity and the lack of a protective effect from MK801, it is log-
ical to conclude that either membrane depolarization occurred
but did not lead to activation of NMDA receptors, or membrane
potentials were resilient to NH4 + interference due to the pres-
ence of K+ channels and Na+ /K+ -ATPase with high substrate
specificity for K+ , in the brain of this catfish. A similar conclu-
sion was drawn by Ip et al. (2005b) on mudskippers, P. schlosseri
and B. boddarti.
212 N.L.J. Wee et al. / Aquatic Toxicology 82 (2007) 204–213
4.4. MSO had a slight protective effect against acute
ammonia toxicity, which appeared unrelated to the
inhibition of GS
MSO is an irreversible inhibitor (Folbergrova, 1964) of GS
in the mammalian system. Administration of MSO could ame-
liorate effects of ammonia toxicity in patients suffering from
hyperammonemic encephalopathy, by reducing the levels of
glutamine synthesized and hence preventing death via the pre-
vention of brain edema (Brusilow, 2002; Butterworth, 2002). In
the case of rats injected intraperitoneally with CH3 COONH4 ,
the protective effects of MSO can also be a result of the pre-
vention of glutamate release and the prevention of activation
of NMDA receptors (Kosenko et al., 1994). In the case of
C. gariepinus injected with a lethal dose of CH3 COONH4
(20 ␮mol g−1 fish), the administration of MSO (100 ␮g g−1 fish)
reduced the mortality rate from 100% to 80% and at the same
time prolonged the time of death significantly from 26.7 min to
47.8 min. Thus, MSO apparently offered some forms of protec-
tion to C. gariepinus confronted with acute ammonia toxicity.
Superficially, these results appear to contradict the earlier con-
clusion that increased synthesis and accumulation of glutamine
was not the major cause of death in C. gariepinus injected
intraperitoneally with a lethal dose of CH3 COONH4 . How-
ever, a detailed analysis of the results reveals that MSO did
not reduce the glutamine level in the brain of the experimental
fish. This phenomenon occurred in spite of the facts that MSO
inhibited (by 45%) GS activity in vitro from the brain of C.
gariepinus, and the prior administration of MSO resulted in a
significant decrease (by 21.5%) in GS activity in the brain of fish
injected intraperitoneally with a lethal dose of CH3 COONH4 .
In the latter fish, the negative effects of a decrease in GS activ-
ity on glutamine formation could have been compensated for
by an increase in glutamate concentration which shifted the
equilibrium in favour of glutamine formation (see discussion
below).
More importantly, a prior injection with MSO resulted unex-
pectedly in a significant decrease (by 38%) in the ammonia
content in the brain of fish injected with a lethal dosage
(20 ␮mol g−1 fish) of CH3 COONH4 . Thus, the protective effects
of MSO on the mortality rate and the time of death of these
experimental fish were unrelated to the inhibition of glutamine
synthesis, but resulted from a slower build up of ammonia in
the brain. This observation is novel, and has not been reported
before for other fish or animal species. How did MSO slow down
the build up of ammonia in the brain of C. garienpinus? This
could not be a result of the brains of the experimental fish being
exposed to a lower level of ammonia, because the plasma ammo-
nia concentrations in fish injected with saline + CH3 COONH4
and those injected with MSO + CH3 COONH4 happened to be
comparable. An original and important observation made in this
study is that the glutamate content in the brain of fish with a
prior injection of MSO was significantly higher than that of
the control fish with a prior injection of saline. These results
give indication for the first time that MSO might have an effect
on glutamate metabolism, and hence activities of GDH, in fish
brains.
4.5. MSO exhibited inhibitory effects on GDH and ALT
activities in vitro
It was reported previously that the administration of MSO
alone induced an increase in GDH activity, in the amination
direction, and a decrease in ALT activity in astrocytes of rat brain
(Subbalakshmi and Murthy, 1983). Subbalakshmi and Murthy
(1983) hypothesized that the GDH reaction might become an
important ammonia detoxification mechanism when the activity
of GS was inhibited by MSO. The glutamate, so formed, would
accumulate instead of being converted to glutamine as observed
by Folbergrova et al. (1969).
While the brain of C. gariepinus injected with MSO + CH3
COONH4 had higher content of glutamate, unlike rat brain
astrocytes, the specific activity of GDH, in the amination
direction, was comparable with that of fish injected with
saline + CH3 COONH4 . However, when tested in vitro, MSO
exhibited quite a prominent inhibitory effect (53%) in vitro on
the activity of GDH from the brain of C. gariepinus. There-
fore, unlike GS, the inhibitory effect of MSO on GDH from
C. gariepinus might be reversible because the GDH activ-
ity from brains of fish injected with MSO + CH3 COONH4
was not significantly different from that of fish injected with
saline + CH3 COONH4 . Superficially, these observations appear
to contradict the fact that MSO lead to an increase in the brain
glutamate content when injected into the fish prior to the injec-
tion with a lethal dose of CH3 COONH4 . However, it can be
reasoned that MSO would simultaneously exert an inhibitory
effect on the deaminating activity of GDH from the brain of
this fish. Taken altogether, these results indicate that MSO sup-
pressed the turnover of glutamate upon its production or release
from protein degradation, resulting in its accumulation in the
brain despite its being siphoned off for glutamine synthesis. In
addition, when tested in vitro, MSO also inhibited ATL activ-
ity (in the direction of alanine degradation) from the brain of C.
gariepinus. Thus, the decrease in rate of ammonia accumulation
in fish injected with MSO + CH3 COONH4 could also result in
part from a decrease in the production of endogenous ammonia
through transdeamination. These results suggest that C. gariepi-
nus could regulate the rate of ammonia production in the brain
when confronted with acute ammonia toxicity, and therefore
efforts should be made in the future to elucidate the regulatory
mechanisms involved. In addition, the effects of MSO alone on
nitrogen metabolism in fish brain should be examined in greater
detail in future studies.
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