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Aquaculture 299 (2010) 149156

Contents lists available at ScienceDirect

Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a q u a - o n l i n e

Metabolic consequences of chronic sublethal ammonia exposure at cellular and

subcellular levels in Nile tilapia brain
Mona M. Hegazi, Zeinab I. Attia, Mohamed A.M. Hegazi , Soha S. Hasanein
Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt

a r t i c l e

i n f o

Article history:
Received 5 June 2009
Received in revised form 17 November 2009
Accepted 23 November 2009
Keywords:
Glutamine
Glutamate
Aspartate
GABA
Alanine
Cytosolic and mitochondrial ammonia
detoxication enzymes

a b s t r a c t
The objective of this study was to elucidate the mechanism of chronic sublethal ammonia exposure in the
brain of Nile tilapia, Oreochromis niloticus, by examining the cytosolic (c) and mitochondrial (m) enzymes
related to ammonia detoxication. The experiment was conducted for 70 days on the juveniles of Nile tilapia
(18 2.1 g) exposed to the total ammonia nitrogen concentration (TAN) of 5 (LL) or 10 (HH) mg L 1 in a
static water system at 26 C. The activities of c-alanine aminotransferase (c-ALT), c-aspartate aminotransferase (c-AST), c-malic enzyme (c-ME), m-malic enzyme (m-ME), glutamate dehydrogenase (GDH) in
the amination direction, and glutamine synthetase (GSase) in sh exposed to one of these sublethal TAN
concentrations were signicantly increased. The increase in the enzyme activity was positively related to the
level of the tested TAN concentration. However, the activity of m-ALT and glutaminase (GLase) was
signicantly decreased, and that of m-AST did not show any signicant changes. On the other hand, in Nile
tilapia exposed to HH, there was a signicant increase in the levels of free amino acids (FAAs), such as
alanine, glutamine, glycine, histidine, serine, and -aminobutyric acid (GABA); however, no alteration in
asparagine was observed, and there was a signicant decrease in the levels of aspartate and glutamate. The
signicant increase in FAAs levels in sh exposed to LL was mainly attributable to alanine, glutamine, and
GABA, and the importance of these metabolic changes in the cytosolic and mitochondrial ALT, AST, and ME
was discussed.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Nile tilapia, Oreochromis niloticus, culture is one of the fastest
growing farming activities. Some of the most important limitations on
the commercial viability of aquaculture are due to water quality
problems, especially the accumulation of ammonia where the sh are
cultured. The total ammonia nitrogen (TAN) in the aquatic environment is the sum of the two forms: unionized ammonia form (NH3)
and ionized ammonium form (NH+
4 ), and the equilibrium exists
mainly between the two forms, as a function of pH and temperature.
The increase in pH is reported to cause increase in the toxic NH3
proportion at the expense of less toxic NH+
4 (Randall and Tsui, 2002).
Chronic exposures of tilapias to increased environmental ammonia
are commonly accompanied with a reduction in the growth
performance. Chronic exposure of sh juveniles to sublethal NH3
concentration resulted in a signicant reduction in the growth rate of
Nile tilapia (El-Shafai et al., 2004) and blue tilapia, Oreochromis aureus
(Hargreaves and Kucuk, 2001). Reduction in the growth performance
was noted in Nile tilapia exposed to 5, 7.5, and 10 mg TAN L- 1 and blue
tilapia exposed to 5 mg TAN L- 1. Hargreaves and Kucuk (2001)

Corresponding author.
E-mail address: m_a_m_hegazi@hotmail.com (M.A.M. Hegazi).
0044-8486/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2009.11.020

reported increased ammonia level in the plasma of blue tilapia

exposed to 5 mg TAN L- 1, and rapidly changed in response to
uctuations in water pH and ammonia concentration.
It has been shown that sh are relatively more tolerable to
ammonia toxicity (Ip et al., 2001a, 2001b, 2004, and 2005). It is clear
that the sh have acquired mechanisms that enable them to resist the
impact of high levels of ammonia in their environment. These
mechanisms are the characteristic of the species and have been
used by the sh to cope with high ammonia exposure. These
mechanisms include: 1) reducing ammonia production by reducing
the rate of amino acid catabolism (e.g. Ip et al., 2001a, Lim et al., 2001
and Tsui et al., 2004), 2) undergoing partial amino acid catabolism
leading to the accumulation of alanine (e.g. Ip et al., 2001a and Chew
et al., 2003), 3) some teleosts detoxify ammonia to urea, through a
complete ornithineurea cycle (OUC) (e.g. Walsh et al., 2004), and 4)
by actively excreting NH+
4 into the water trapped between the
secondary lamellae of the gills (e.g. Randall et al., 1999). In addition,
there are some reports that have demonstrated that sh can detoxify
ammonia to glutamine via glutamine synthetase (GSase) activation in
their brain (e.g. Mommsen and Walsh, 1992; Hernndez et al., 1999;
Ip et al., 2001a; Wicks and Randall, 2002; Saha et al., 2002 and Wee
et al., 2007).
However, there are no data available on Nile tilapia with respect to
this criterion during ammonia loading. Nevertheless, there are data

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M.M. Hegazi et al. / Aquaculture 299 (2010) 149156

related to how Nile tilapia excretes urea during ammonia loading. Nile
tilapia is ammonotelic, and urea-N accounts for only 19% of the total
waste-N at 30 C (Alsop et al., 1999). McKenzie et al. (1999) showed
that Nile tilapia does not possess a functional OUC, and that carbamoyl
phosphate synthetase III activity could not be detected in its liver,
even after being injected twice with 5 mL kg 1 of 200 mM of NH4Cl.
The sh was observed to increase its urea excretion following
exposure to elevated water ammonia, not associated with the
activation of the enzymes of OUC, and urea was mainly produced by
uricolysis (Wright, 1993). It has been suggested that in Nile tilapia,
increased water ammonia may increase the activity of the uricolytic
enzyme, allantoicase, although this change in activity may not be
directly related to the changes in the plasma urea levels or urea
movements (Wright, 1993).
The increased total activity of alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) in the brain of the sh species
exposed to sublethal levels of ammonia has been demonstrated (e.g.
Mommsen and Walsh, 1992; Hernndez et al., 1999; Wicks and
Randall, 2002; Saha et al., 2002 and Wee et al., 2007). In addition,
activated GSase consumes more glutamate produced via amination of
glutamate dehydrogenase (GDH), thus preventing its accumulation.
These enzymes are used in ammonia detoxication. However, there
are no available data demonstrating the effect of ammonia exposure
on the activity of the cytosolic (c) and mitochondrial (m) ALT, AST,
and malic enzyme (ME) in sh brain. Until recently, the measurement
of the enzymes c- and m-ALT, AST, and ME isoforms at the subcellular
compartments was not applied in the evaluation of the sh's capacity
to cope with environmental NH3 loading. The early enzymatic kinetic
study by DeRosa and Swick (1975) on the mammalian animal model
revealed that c-ALT, when compared with m-ALT, seems to have
different functional properties. The kinetic parameters of the two
isoforms suggest that in vivo, m-ALT is involved in the conversion of
alanine to pyruvate, while c-ALT is mainly involved in the formation of
alanine from pyruvate. Recently, based on the biochemical studies, it
was proposed that the two ALT isoforms in sh show different kinetic
parameters, similar to that of the mammal's patterns (Metn et al.,
2004 and Srivastava et al., 2004). The two ALT isoforms have been
cloned by Anemaet et al. (2008) from the liver of sea bream, Sparus
aurata, and their biochemical characterization ascertained their
different functions. On the other hand, both c-ASE and m-AST
isoforms have been observed to act as a key in providing the substrate
for the malate aspartate shuttle pathway (McKenna et al., 2006b).
Accordingly, the activity of c-ASE and m-AST outside and inside the
mitochondrial wall must be determined to examine the malate
aspartate shuttle pathway. Determination of the activity of c- and mALT and AST isoforms individually may prevent obscure result owing
to the differences in their functions.
In spite of the recent advances in research on the mechanisms used
by some sh species to cope with high environmental ammonia
exposure, there has been limited knowledge in this respect with
regard to Nile tilapia. To date, such mechanisms have not observed in
Nile tilapia. Thus, the aim of this study was to throw some light on
how Nile tilapia defends against chronic sublethal TAN concentrations
(5 or 10 mg L 1 at 26 C). The present investigation is the rst trial in
this eld to study the enzymes at cellular and subcellular levels in sh
exposed to chronic sublethal ammonia concentrations. This was
carried out by evaluating the activity of the cytosolic and mitochondrial enzyme isoforms that take part in ammonia detoxication in Nile
tilapia brain. The study of enzyme isoforms, which may be more
informative, was not taken into consideration in the previous
investigations. Hence, a comprehensive chronic toxicity trial was
conducted consecutively for 70 days using a static water system to
study the effect of sublethal ammonia exposure on sh juveniles. The
activity of GDH, GSase, GLase, c-ALT, m-ALT, c-AST, m-AST, c-ME, and
m-ME was evaluated. We tried to shed light on the mechanism(s) that
show how the Nile tilapia can cope with chronic NH3 loading. We

examined whether 1) ammonia would be detoxied to glutamine,

leading to its accumulation in the brain and 2) free amino acids (FAAs)
would be accumulated in the brain. The accumulation of a
neurotransmitter FAA, such as GABA and glycine, in sh brain was
found to be an indication of neurological disturbances in the brain.
Subsequently, the efciency of 1) malate aspartate shuttle, as a major
processing energy tool between cytosol and mitochondria and 2)
pyruvate recycling, as a pathway for partial and/or complete oxidation
of glutamate leading to alanine accumulation, was studied.
2. Materials and methods
2.1. Experimental system conditioning
In early October 2007, juveniles of Nile tilapia from the same
parental stock were transported from the sh-hatching pond in Fowa
city (Kafr El-Sheikh Governorate, Egypt) to our laboratory, in
oxygenated cellophane bags. On arrival, sh of similar sizes (18
2.1 g) were distributed into 30 glass aquaria, each with a rearing
volume 33 L (2 sh in each) and rearing density of approximately
1.1 g L 1 in dechlorinated city tap water. The sh density was
reduced at the beginning of the experimental time to prevent the nal
densities from exceeding the recommended threshold. The upper
rearing density limit of the sh for toxicity testing was 2.5 g L 1
(American Public Health Association, 1998). The nal rearing density
at the end of the experiment was 2.42 g L 1. The aquaria were
supplied with continuous aeration through sponge biological lters.
The dissolved oxygen level was maintained at around 8085% of
saturation. The sh were exposed to normal photoperiod from 10 h
light to 14 h dark in the laboratory. The daily feeding ration of
commercial sh pellets diet was 3% of the sh's body weight, during
the experimental time (25.2% protein, 5% lipids, and 5.7% bers, with a
total energy of 2505 kcal kg 1). For 2 weeks, the sh were allowed to
adapt to the pre-selected experimental conditions. For 10 days, the
temperature was steadily increased from the eld temperature that
uctuated around 18 C, to 26 C by using highly precise thermostatic
heaters. Subsequently, the sh were acclimated for the next 4 weeks
to 26 C, the optimal temperature of the Nile tilapia for maximum
growth (Mishrigi and Kubo, 1978).
2.2. Experimental ammonia adjustment and exposure
The sh of the aquaria were classied into three subgroups,
including those in each subgroup (10 aquaria). The rst 10 aquaria
were left as controls and each of the other two subgroups received one
of the two nominal TAN of 5 or 10 mg L 1. Ammonium chloride
(NH4Cl, 99.5% purity) stock solution (50 g L 1) was used as a source
of TAN concentrations. Subsequently, the experiments were carried
out over a period of 70 consecutive days. The pH values of different
aquaria were registered daily. Every day, 75% of the aquaria's water
was replaced with dechlorinated tap water, preheated to 26 C.
El-Shafai et al. (2004) used the selected sublethal TAN nominal
concentrations of 5 or 10 mg L 1 for 75 days, and demonstrated the
reduction in the growth performance of the juvenile Nile tilapia. The
pH values of the aquaria were measured using a pH meter (Hanna
Instruments, HI9024). TAN concentration in the aquaria was measured by Nesslerization (Gentzkow and Masen, 1942), using NH4Cl as
a standard. The ammonia and pH levels were observed to decline
slowly owing to ammonia volatilization and nitrication. The water
parameters were measured and adjusted by adding NH4Cl solution
daily. The values of NH3 were calculated from TAN, pH, and
temperature of the aquaria, according to the general equations of
Albert (1973). The calculated concentrations of NH3 in the aquaria
were 0.059 (controls), 0.185 (LL), and 0.575 (HH) mg L 1 at pH 7.6
0.05, 7.8 0.06, and 8.0 0.08, respectively, at 26 C.

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2.3. Biochemical assays

2.3.1. Preparation of tissue homogenates for enzyme assays
At the end of each experiment, the sh was killed by a sharp blow
on the head and the whole brain was quickly excised, washed in
isoosmolar NaCl saline to remove blood, and weighted. The
isoosmolar buffer (B1) was used for the extraction of cytosolic
enzyme fraction, while the hypoosmolar buffer (B2) was used for the
extraction of the cytosolic and mitochondrial enzyme fraction,
according to the procedure described by Moon and Ouellet (1979),
with some modications. The B1 buffer contained 20 mmol L 1
HEPES, pH 7.6, 1 mmol L 1 EDTA, 30 mmol L 1 -mercaptoethanol,
and 250 mmol L 1 sucrose. On the other hand, the B2 buffer
contained 20 mmol L 1 HEPES, pH 7.6, 1 mmol L 1 EDTA, 1% triton
X-100, and 30 mmol L 1 -mercaptoethanol.
Two brain samples were pooled and homogenized (1:2 w:v) in
cold buffer B1 (4 C) using an all-glass manual homogenizer
immersed in ice. About 200 L of the resulting homogenate was
delivered to a centrifuge tube and diluted using the B1 buffer to bring
the dilution to 1:10 w:v. To another centrifuge tube, an equal volume
from the resulting homogenate was also delivered and diluted using
the B2 buffer to bring the dilution to 1:10 w:v. The contents of each
tube were mixed thoroughly. Thus, the isoosmolar and hypoosmolar
medium for the homogenate, adjusted by the B1 and B2 buffer,
respectively, can be obtained. The homogenate of B1 buffer was
centrifuged using a cooling centrifuge at 12,000 g for 15 min at 4 C.
The yielded high-speed supernatant of B1 was decanted and used
immediately to examine the cytosolic fraction of ALT, AST, and ME.
The homogenate of B2 buffer was frozenthawed three times for the
complete disruption of mitochondria (Salach, 1978). The homogenate
was then centrifuged using a cooling centrifuge at 6000 g for 15 min
at 4 C. The resulting supernatant containing the cytosolic and
mitochondrial fractions was decanted for immediate enzyme assays.
The B2 fraction contained the enzymes GDH, GSase, GLase, and total cand m-ALT, AST, and ME. The supernatants were carefully collected,
avoiding contamination with the upper lipid layer at the top of the
tubes. The mitochondrial ALT, AST, and ME activities represent the
difference between the enzyme activities in the two fractions. To test
the mitochondrial-residue contaminations in the cytosolic fraction of
B1, the activity of GDH was assayed in the cytosolic fraction. In the
procedure described by Moon and Ouellet (1979), the intact
mitochondria was separated and repeatedly washed and centrifuged
several times using the isoosmolar buffer, to ensure that there is no
contamination in the cytosolic fraction. However, during washing, a
considerable quantity of mitochondria was lost. Thus, the remaining
mitochondrial content after washing cannot be considered as the
actual content of the tissue. Hence, an obligate chemical protein
quantication of the washed mitochondria disrupted in the hypoosmolar buffer was required for the method described by Moon and
Ouellet. Accordingly, the enzyme activity of the mitochondria must be
presented with reference to the protein concentration. The modication presented in this paper demonstrates the actual content of the
enzymes activity per wet weight tissue. Our modication made the
procedure easier in two ways: 1) evidences ascertained that no
contamination occurred between the mitochondrial and cytosolic
fractions and 2) the results of the mitochondrial and cytosolic enzyme
activity were presented with reference to the wet mass tissue, thus
facilitating the computation of the activity of each fraction.
2.3.2. Enzyme assays
The activity of the enzymes ALT, AST, ME, and GDH were assayed
kinetically based on the method described by Foster and Moon (1986).
The activity was assessed by monitoring the absorbance at 340 nm
(Ex = 6.22 mmol L 1 1 1 cm2) using a thermostated UV/VIS Spectrophotometer (JENWAY 6505 U.K) at 25 C. The concentration of each
enzyme and substrate was adjusted to provide a linear detectable

151

increase in the extinction to give the maximal specic activity. The

activity was corrected for any nonspecic activity in the absence of
substrate. The reaction was started by the addition of an appropriate
amount of cytosolic or cytosolic and mitochondrial fraction. The
maximal specic activity was expressed in mol min 1 g 1 wet
weight. The analytical grade chemicals used were purchased from
Sigma Chemical Co. (St. Louis, MO).
ALT (E.C. 2. 6. 1. 2.) was assayed in the reaction mixture containing
65 mmol L 1 imidazolHCl buffer (pH 7.4), 200 mmol L 1 L-alanine,
12 mmol L 1 -ketoglutarate (-KG), 0.15 mmol L 1 NADH,
0.025 mmol L 1 pyridoxal phosphate, 1 mmol L 1 EDTA, and 12 U/mL
lactate dehydrogenase (LDH, E.C.1.1.1.27). AST (E.C.2.6.1.1) was assayed
in the reaction mixture containing 65 mmol L 1 imidazolHCl buffer
(pH 7.4), 50 mmol L 1 L-aspartate, 10 mmol L 1 -KG, 0.15 mmol L 1
NADH, 0.025 mmol L 1 pyridoxal phosphate, 1 mmol L 1 EDTA, and
8 U/mL malate dehydrogenase (MDH, E.C.1.1.1.37). ME (E.C.1.1.1.40)
was assayed in the reaction mixture containing 5 mmol L 1 malate,
0.4 mmol L 1 NADP, and 5 mmol L 1 MnCl2. GDH (E.C.1.4.1.2) reductive amination reaction was assayed in the reaction mixture containing
65 mmol L 1 imidazolHCl buffer (pH 7.4), 5 mmol L 1 -KG,
1 mmol L 1 ADP, 0.15 mmol L 1 NADH, 100 mmol L 1 CH3COONH4,
and 1 mmol L 1 EDTA.
GSase (E.C. 6.3.1.2 (was assayed according to the method
described by Shankar and Anderson (1985). The formation of glutamylhydroxymate was determined at 500 nm. Furthermore,
phosphate-activated GLase (E.C. 3.5.1.2) was assayed according to
the method described by Curthoys and Weiss (1974).
2.3.3. HPLC assay for amino acids
Two brain samples were pooled and homogenized in 75% aqueous
methanol (HPLC grade) of 1/10 (w/v), centrifuged at 3000 g
for10 min, and the supernatant was retained. The supernatant was
taken immediately and the trace elements and lipids were extracted
using solid-phase extraction Chromabond column NH2 phase (Cat.
No.730031). The supernatant was used for assaying the FAA levels,
such as alanine, aspartate, asparagine, glutamate, glutamine, glycine,
histidine, serine, and GABA, by employing PerkinElmer HPLC consisting of quaternary pump, a column oven, Rheodyne injector, and 20-L
loop UV variable wavelength detector, according to the method
described by Heinrikson and Meredith (1984).
2.4. Statistical analysis
One-way analysis of variance, ANOVA, was used to analyze the
data of the enzyme activities, to check the effect of sublethal NH3
concentrations. The signicance of difference owing to the effects of
NH3 concentrations was evaluated using multiple comparison
Dunnett test (compare all vs. controls), using a computer program
(GraphPad InStat Software, Inc.).
3. Results
The temperature was the same in all aquaria (26 0.5 C). The
values of water quality in the treatment aquaria are presented in
Table 1. The DO was 8085% in all aquaria. The calculated concentrations of NH3 in aquaria in mg L 1 were 0.059 (controls) 0.185 (LL)
and 0.575 (HH) at pH 7.6 0.05, 7.8 0.06 and 8.0 0.08 respectively. No signicant differences in the total hardness and total
alkalinity were detected between the treatment aquaria.
3.1. Enzyme activity
Each reading of the enzyme activities and FAA represents the
mean SD of ten samples. Each specic sample for the investigation
of enzyme activity or FAAs contained two pooled brains.

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Table 1
Water quality in sh aquaria during the experimental period of chronic ammonia
toxicity at 26 C.
Parameter

Controls

5 mg TAN L 1

10 mg TAN L 1

pH
DO saturation%
NH3 mg N L 1
Nitrite mg N L 1
Nitrate mg N L 1
Total hardness mgCaCO3 L 1
Total alkalinity

7.6 0.05
8085
0.059
0.045 0.005
0.07 0.006
120 1
127 3

7.8 0.06
8085
0.185
0.042 0.006
0.085 0.007
120 1
128 4

8.0 0.08
8085
0.575
0.45 0.006
0.092 0.008
120 1
129 6

NH3 concentration was calculated as a function of nominal total ammonianitrogen

concentration (5 and 10 mg L 1), pH, and temperature during the experimental
periods.

The effects of the two chronic sublethal concentrations of ammonia

LL or HH on the activity of key enzymes related to ammonia
detoxication of Nile tilapia brain, when compared with the controls
are presented in Fig. 1(A, B, C, and D). There were signicant alterations
in the activity of brain enzymes, namely, c-ALT, c-AST, c-ME, m-ME, GDH
(amination), GSase, and GLase of the sh brain with respect to LL or HH
exposures. The activity of c-ALT was signicantly increased by 85%
(P b 0.05) in sh exposed to LL, and by more than 2-fold (P b 0.01) in sh
exposed to HH (Fig. 1A). The activity of c-AST was signicantly increased
by 1.4-fold (P b 0.05) in sh exposed to LL and by more than 2.4-fold
(P b 0.01) in sh exposed to HH (Fig. 1A). The activity of m-ALT was
signicantly decreased by 21.25% (P b 0.05) in sh exposed to LL and by
25.4% (P b 0.01) in sh exposed to HH (Fig. 1A). However, the activity of
m-AST was not signicantly changed in sh exposed to LL or HH
(Fig. 1A). The activity of c-ME was signicantly increased by 38.5%

(P b 0.05) in sh exposed to LL and by 25.4% (P b 0.01) in sh exposed to

HH. The increase in the activity of m-ME was more detectable than that
of c-ME (Fig. 1B). The activity of m-ME was signicantly increased by
63.6% (P b 0.05) in sh exposed to LL and by more than 2-fold (P b 0.01)
in sh exposed to HH.
The increase in the activities of the enzymes GDH (amination
direction) and GSase (Fig. 1C) was more prominent than those listed
earlier. The activity of GDH was signicantly increased by 82.6%
(P b 0.01) in sh exposed to LL and by 2.2-fold (P b 0.01) in sh
exposed to HH (Fig. 1C). The activity of GSase was signicantly
increased by 2.4-fold and 3.3-fold (P b 0.01) in sh exposed to LL and
HH, respectively (Fig. 1C). The activity of GLase (Fig. 1D) was
signicantly decreased by about 50% (P b 0.01) in sh exposed to LL
and HH, respectively.

3.2. Free amino acids

The effects of the two chronic sublethal concentrations LL or HH on
FAAs of Nile tilapia brain, when compared with the controls, are
presented in Fig. 2(A, B, and C).
Furthermore, there was a signicant increase (P b 0.01) in the
levels of glutamine by 42.8 and 61% in sh exposed to LL and HH,
respectively. The levels of glutamate remained unchanged in sh
exposed to LL; however, it signicantly decreased (P b 0.05) by 18% in
sh exposed to HH (Fig. 2A).
Aspartate levels signicantly decreased (P b 0.01) by 14% and 30%
in sh exposed to LL and HH, respectively. On the other hand, the
glycine levels were signicantly increased by 28% in sh exposed to
LL. Furthermore, there was a signicant increase (P b 0.05) in the

Fig. 1. (A, B, C, and D): Activity of amino acid related enzymes in the whole brain tissues of Nile tilapia controls (1), exposed to LL (2), and HH (3) concentrations of NH3 for 70 days at
26 C. Each reading represents Mean SD of 10 samples. The difference was signicant at P 0.05 and P 0.01.

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M.M. Hegazi et al. / Aquaculture 299 (2010) 149156

Fig. 2. (A, B and C): Amino acids concentration in the whole brain tissues of Nile tilapia
exposed to LL and HH concentrations of NH3 for 70 days at 26 C. Each reading
represents Mean SD of 10 samples. The difference was signicant at P 0.05 and
P 0.01.

GABA levels by 65.6% and 3-fold in sh exposed to LL and HH,

respectively (Fig. 2B).
There was a signicant increase in the levels of alanine by 37.5%
and 2.1-fold; histidine by 2.2-fold and 3.2-fold; and serine by 53.8 and
70%, in sh exposed to LL and HH, respectively. However, asparagine
levels remained unchanged in sh exposed to LL or HH (Fig. 2C). The
signicant increase in FAA levels in sh exposed to LL can primarily be
attributed to alanine, glutamine, histidine, and GABA.
4. Discussion
4.1. Metabolic pathways
The activity of GDH in amination direction increased in the brain of
Nile tilapia exposed to LL. However, the increase in the activity was
more prominent in sh exposed to HH. In the vertebrate cells, GDH,
primarily associated with the mitochondrial matrix, has a regulatory

153

function in cellular metabolism by controlling the levels of ammonia

and glutamate. Synthesis of glutamate by assimilating ammonia with
-KG with the help of GDH, during the process of amination direction
plays a role in the detoxication of ammonia produced endogenously
from the catabolism of amino acids and proteins in various tissues
including the brain (Cooper and Plum, 1987). The increase in the GDH
activity during amination direction may aid in converting the
accumulated ammonia to glutamate, preceded through GSase to
glutamine detoxifying more ammonia. The activity of GSase was
found to increase in the brain of Nile tilapia exposed to LL, and the
increase was more in sh exposed to HH. The normal values of GSase
activity in the brain of Nile tilapia in the present experiment was
slightly lower than those previously quantied by Mommsen et al.
(2003). This may be related to the difference in the sh weight, where
the sh weight ranged from 80 to 200 g in Mommsen's experiment.
Brain GSase is an essential tool for the removal of the toxic effects of
ammonia. In the brain of all the vertebrates, astrocytic GSase
detoxies ammonia during the conversion of glutamate to glutamine
at the expense of ATP, a process that is also one of the steps in the
recycling of the neurotransmitter, glutamate, taken up from the
synaptic cleft through a glutaminase/glutamine synthetase cycle
(Cooper, 2001). The increased GDH and GSase activities in the brain
tissues are related to the ammonia detoxication mechanism that
protects the brain of both mammals (Cooper and Plum, 1987) and sh
(e.g. Mommsen and Walsh, 1992; Hernndez et al., 1999; Wicks and
Randall, 2002; Saha et al., 2002 and Wee et al., 2007).
The increased c-ALT activity in the brain of Nile tilapia may favor
L-alanine formation from pyruvate and trap NH3. It was hypothesized as
being mainly involved in the conversion of pyruvate to L-alanine. This
may be ascertained by the increased L-alanine level in the Nile tilapia
brain tissues (Fig. 2C). On the other hand, mitochondrial ALT exhibited
higher afnity for L-alanine and was considered to be mainly involved in
the reverse reaction (Metn et al., 2004 and Srivastava). The decreased
m-ALT activity may indicate a decrease in pyruvate formation from
alanine and reduced NH3 formation. The increase in c-ALT activity in
combination with the decrease in m-ALT activity may be an adopted
mechanism to reduce NH3 production when exposed to high NH3
concentrations. Alanine can be formed through the pathway catalyzed
by c-ALT, such as glutamate + pyruvate -KG + alanine. The resultant -KG enters the Krebs cycle, and undergoes partial oxidation to
malate yielding 6 ATP (Fig. 4). Further, an increased rate of pyruvate
from malate may be expected, as indicated by increased c- and m-ME
activity.
Malate is subsequently catalyzed by ME and converted to pyruvate
(Fig. 4), yielding 1 NADPH. This enzyme reaction is potentially
reversible, but at the physiological concentrations of malate and
pyruvate, more than 95% of malate is transferred to pyruvate
(Kimmich et al., 2002). Pyruvate recycling is a pathway for partial
and/or complete oxidation of glutamate. Olstad et al. (2007)
concluded that pyruvate recycling is evident in the neurons. Though
c-ME is a predominantly glial enzyme, neurons in vivo express m-ME,
through which pyruvate recycling can occur (Cruz et al., 1998). Malate
can be converted to pyruvate via either c-ME and/or m-ME in the
brain (McKenna et al., 1990). This pathway is essential for the partial
and/or complete oxidation of glutamate (Tildon and Zielke, 1988).
Accordingly, the increased activity of c-ALT, c-ME, and m-ME may
contribute in lowering the glutamate levels in the neurons. It has been
well established that glutamate is removed from the synaptic cleft in
astrocytes through the following mechanisms: 1) conversion to
glutamine, 2) oxidation for energy via the TCA cycle after either
oxidative deamination via GDH, or transamination via AST, and 3) in
the biosynthesis of glutathione and proteins, and/or in the purine
nucleotide pathway (Cooper et al., 1983).
The present increase in the activity of c-ALT, c-ME, and m-ME with
the decrease in the activity of m-ALT can be considered as a metabolic
adaptation mechanism that contribute to the 1) reduction in NH3

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M.M. Hegazi et al. / Aquaculture 299 (2010) 149156

production to cope with high internal NH3; 2) reduction in the

excitatory neurotransmitter glutamate; 3) release of energy from
partial oxidation of amino acids without great excessive NH3
liberation. The present pattern of enzyme activity might be emphasizing this criterion as an evolutionary mechanism to cope with NH3
loading in the brain of Nile tilapia. The later mechanisms and
metabolic pathways hypothesized for glutamate and glutamine
synthesis are depicted in Figs. 3 and 4.
The increase in c-AST activity and the decrease in m-AST activity
may signify a concept other than that suggested by a freely reversible
transaminase. The two distinct enzymes, c-AST and m-AST, were
isolated from the sh tissues and its properties were studied (Petrovic
et al., 1996 and 1999). Both the cytosolic and mitochondrial isoforms
of this enzyme were observed to act as a key that provides the
substrate for the malate aspartate shuttle pathway (McKenna et al.,
2006b). This pathway was found to be instrumental in the
transportation of crucial intermediates across the mitochondrial
membrane, and thereby channeling the metabolites into its general
metabolic pathways and transferring the reducing equivalents from
the cytosol into the mitochondria (Srivastava et al., 2004) to maintain
the energetics of the brain. The enzyme m-AST has a number of
specic roles in astrocytes and neurons in the brain (McKenna et al.,
2006a and references therein). This enzyme aids in the entry of
glutamate into the TCA cycle, as well as its re-synthesis from TCA cycle
intermediates. In addition, m-AST helps in the synthesis of the
neurotransmitter, glutamate, in the brain. The observed decrease in
m-AST may indicate a shortage of malate aspartate shuttle capacity,
signifying a decreased energy budget in the brain tissue exposed to
ammonia. It has been shown that NH3 could induce depletion in the
brain ATP in vivo (Kosenko et al., 1994). However, the increased c-AST
activity in the brain tissue exposed to ammonia is elusory.
4.2. Free amino acids
There was a signicant increase in most of the FAA levels in the
brain of Nile tilapia exposed to LL and HH concentrations. The
accumulation of these amino acids in brain seems to be the classical
mechanism that ameliorates ammonia toxicity. It was suggested that
such a strategy may be adopted by sh to avoid NH3 toxicity during
high ammonia loading (Mommsen and Walsh, 1992; Hernndez et al.,
1999; Wicks and Randall, 2002; Saha et al., 2002; Wee et al., 2007).
The NH3 molecule crosses the bloodbrain barrier much more readily
(Felipo and Butterworth, 2002). Many sh detoxify NH3 to glutamine

Fig. 3. The metabolic pathways hypothesized for glutamate and glutamine formation.
The heavy arrow donated to the direction which the enzyme activity favors.

Fig. 4. The metabolic pathways hypothesized for partial oxidation of -ketoglutamate

through TCA cycle. The heavy arrow donated to the direction which the enzyme activity
favors.

when exposed to elevated environmental ammonia. There was a

signicant increase in glutamine levels in the brain of Nile tilapia
exposed to LL. However, the increase was prominent when the sh
were exposed to HH concentrations. Furthermore, the brain glutamine levels showed a positive correlation with ambient ammonia
exposure in rainbow trout, Oncorhynchus mykiss (Wicks and Randall,
2002), mudskippers, Periophthalmodon schlosseri and Boleophthalmus
boddaerti (Ip et al., 2001a; 2005), and toadsh, Opsanus beta (Veauvy
et al., 2005). During aerial exposure, ammonia also detoxied to
glutamine in sleeper, Bostrichthys sinensis (Ip et al., 2001b), marble
goby, Oxyeleotris marmoratus (Jow et al., 1999), air-breathing catsh,
Clarias batrachus (Saha et al., 2002), and African sharptooth catsh,
Clarias gariepinus (Wee et al., 2007). Astrocyte cell swelling has
indeed been implicated in a wide variety of disorders affecting the
brain. In human hepatic encephalopathy, for instance, the enhanced
NH3 concentration forces the formation and cellular accumulation of
glutamine, leading to astrocyte cell swelling (Cooper, 2001).
The exposure of Nile tilapia brain to HH concentrations leads to
lower concentration of aspartate and glutamate, while LL concentrations are observed to lower the concentration of aspartate only
(Fig. 2A). The higher concentration of glycine was signicant after HH
exposure. GABA showed a signicant increase in sh exposed to LL
and HH concentrations. These increases in GABA were positively
correlated to the NH3-exposure level. Aspartate and glutamate are the
essential components of the malateaspartate shuttle, and depletion
of brain aspartate and glutamate concentrations may interfere with
this shuttle system and adversely affect the brain energy metabolism
(see McKenna et al., 2006a for review). GABA and glycine are
considered to be the major inhibitory neurotransmitters in the central
nervous system (CNS) of the vertebrates. GABA released from
GABAergic neurons, form approximately 30% of all central synapses
(see Basile, 2002 for review). Impairment of astrocytic function may
partly be accompanied by changes in the uptake and release of GABA.
Elevation in ammonia concentrations suppresses the astrocytic GABA
uptake (Bender and Norenberg, 2000) and/or increases the neuronal
and/or astrocytic GABA release (Wysmyk et al., 1992). The increased
levels of GABA in the brain of Nile tilapia exposed to LL or HH
concentrations may be related to impairment in astrocytic function.
On the other hand, glycine encephalopathy is a disorder of amino acid
metabolism in which large concentrations of glycine accumulate in all
the body tissues, including the CNS. Damage caused by increased
amounts of glycine in CNS is responsible for mental retardation,
seizures, etc., which are the characteristics of glycine encephalopathy
(Morrison et al., 2006). When the glycine cleavage enzyme, a
mitochondrial decarboxylase, is defective, excess glycine can reach
toxic levels in the body's organs and tissues. The defect causes the
accumulation of large amounts of glycine in all the body tissues
including the CNS (Yang and Svensson, 2008). From these observations, it can be concluded that there is a damage caused by alterations
in the amounts of amino acids, such as glutamate, aspartate,
glutamine, GABA, and glycine, especially when the CNS of Nile tilapia
is exposed to HH.

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M.M. Hegazi et al. / Aquaculture 299 (2010) 149156

5. Conclusion
The regulation of c-ALT and/or m-ALT, AST, and ME, in addition to
GDH and GSase, cannot be ruled out in Nile tilapia exposed to chronic
sublethal ammonia stress. Most FAA concentrations were signicantly
increased in the brain of Nile tilapia under this condition. In addition,
the possible conversion of ammonia to different FAAs by the Nile tilapia
was looked upon as another potential mechanism of survival at a high
concentration of ambient ammonia. The accumulation of FAAs in the
brain of Nile tilapia may be due to the increase in the amino acid
synthesis from ammonia and -keto acids catalyzed by c-ALT and c-AST
and other aminotransferases, while that of GDH and GSase may be
owing to ammonia loading. The high ammonia loading may push the
reactions toward amino acids synthesis and their accumulation.
Reduction in the rates of catabolism of FAAs might be a valuable
adaptation for a sh in ammonia loading. The partial oxidation of FAAs
can also be included, as indicated by the increase in the activity of c-ME
and m-ME observed in this study, which are links between malate from
Krebs cycle and pyruvate production. These functional changes seem to
be of physiological signicance and also provide preliminary evidence
for subtle changes in the enzyme as a hallmark metabolic adaptation in
Nile tilapia exposed to chronic sublethal ammonia concentrations.
However, many neurological disorders that may occur owing to
glutamine and/or glutamate accumulation cannot be ruled out.
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