1. Neurochem Res (2008) 33:103–113
DOI 10.1007/s11064-007-9422-x
ORIGINAL PAPER
Acute and Chronic Hyperammonemia Modulate Antioxidant
Enzymes Differently in Cerebral Cortex and Cerebellum
Santosh Singh Æ Raj K. Koiri Æ Surendra Kumar Trigun
Accepted: 18 June 2007 / Published online: 4 August 2007
Ó Springer Science+Business Media, LLC 2007
Abstract Studies on acute hyperammonemic models stress. This is supported by ~ 2- and 3-times increases in the
suggest a role of oxidative stress in neuropathology of level of lipid peroxidation in cerebellum during chronic and
ammonia toxicity. Mostly, a low grade chronic type acute HA respectively, however, with no change in the
hyperammonemia (HA) prevails in patients with liver cortex due to chronic HA.
diseases and causes derangements mainly in cerebellum
associated functions. To understand whether cerebellum Keywords Hyperammonemia Á Ammonia neurotoxicity Á
responds differently than other brain regions to chronic type Antioxidant enzymes Á Oxidative stress Á Cerebral cortex Á
HA with respect to oxidative stress, this article compares Cerebellum
active levels of all the antioxidant enzymes vis a vis extent
of oxidative damage in cerebral cortex and cerebellum of
rats with acute and chronic HA induced by intra-peritoneal Introduction
injection of ammonium acetate (successive doses of
10 · 103 & 8 · 103 lmol/kg b.w. at 30 min interval for Hepatic encephalopathy (HE) is a serious nervous system
acute and 8 · 103 lmol/kg b.w. daily up to 3 days for disorder developed due to increased ammonia level in brain
chronic HA). As compared to the respective control sets, resulting from liver dysfunction. This is of great concern
cerebral cortex of acute HA rats showed significant decline because a number of liver disorders like viral hepatitis,
(P < 0.01–0.001) in the levels of superoxide dismutase liver intoxication, alcoholism and inborn errors of urea
(SOD), catalase and glutathione peroxidase (GPx) but with cycle are associated with different grades of hyperammo-
no change in glutathione reductase (GR). In cerebellum of nemic conditions in the patients [1]. It has been reported
acute HA rats, SOD, catalase and GR though declined that acute ammonia exposure of brain cells causes
significantly, GPx level was found to be stable. Contrary to dysfunction of multiple neurotransmitter system [1, 2] and
this, during chronic HA, levels of SOD, catalase and GPx glutamate & ammonia mediated excitotoxicity of neurons
increased significantly in cerebral cortex, however, with a [3]. At down stream level, defects in brain bioenergetics [4]
significant decline in the levels of SOD and GPx in cere- and mitochondrial dysfunction mediated oxidative stress
bellum. The results suggest that most of the antioxidant [5, 6] are considered to play important roles in patho-
enzymes decline during acute HA in both the brain regions. physiology of HE. Moreover, most of the evidences for a
However, chronic HA induces adaptive changes, with role of oxidative stress in ammonia neurotoxicity have
respect to the critical antioxidant enzymes, in cerebral cor- been derived either from cell culture studies [6, 7] and/or
tex and renders cerebellum susceptible to the oxidative from acute hyperammonemic animal models [8–10].
Nonetheless, low grade chronic hyperammonemic condi-
tion is more prevalent in the patients suffering from viral
hepatitis and liver dysfunction due to alcoholism and long
S. Singh Á R. K. Koiri Á S. K. Trigun (&)
term drug abuse. Therefore, it is important to understand
Biochemistry & Molecular Biology Laboratory, Department of
Zoology, Banaras Hindu University, Varanasi 221005, India how chronic HA affects cellular antioxidant defense
e-mail: sktrigun@sify.com; sktrigun@bhu.ac.in mechanisms in susceptible brain regions.
123

2. 104 Neurochem Res (2008) 33:103–113
There are some reports on the role of oxidative stress in Experimental procedure
chronic hyperammonemic models also; however, most of
them are focused to the hyperammonemia (HA) dependent Animal and chemicals
impairment of NMDA receptor activity [1] via alterations
in glutamate-NO-cGMP pathway [11, 12]. In addition, Male adult albino rats weighing 100–120 g were main-
chronic HA has been found to induce adaptive changes tained in an animal house as per the recommendations from
in brain energy and ammonia metabolites, which are institutional ethical committee for the care and use of
altered otherwise during acute ammonia intoxication [13]. laboratory animals.
Increases in the levels of ammonia, glutamate and mito- All chemicals used were of analytical grade or of the best
chondrial NAD/NADH ratio in chronic HA models [13] quality supplied by E-Merk, Glaxo and SRL (INDIA).
hint for a mitochiondrial dysfunction and implication of Acrylamide, N N-methylene bis acrylamide, Coomassie
oxygen free radicals in the pathophysiology of chronic type Brilliant Blue R-250 (CBB), TEMED (N N N N-tetrameth-
HE also. Nonetheless, information is scarce on implication ylethylene diamine) and Phenyl methyl sulphonyl fluoride
of antioxidant enzyme system during chronic HA in animal (PMSF) were purchased from Sigma Chemical Co., USA.
models.
Primary level neuropathology of HE, like motor Experimental design
disturbances, expressionless face, rigid muscle tone, tremor
etc, is common with the low-grade chronic type HE Acute and chronic HA in rats were induced by intraperi-
patients [14, 15] and these functions are mainly associated toneal injection of ammonium acetate prepared in physio-
with the derangements in motor activities of cerebellum. logical saline (0.9% NaCl). As described earlier [24], for
Thus, it is likely that cerebellum responds differently to acute HA, first 10 · 103 lmol/kg b.w. of ammonium
HA than the other brain regions. Differential susceptibility acetate was administered to the rats followed by a second
of cerebellum and cerebral cortex with respect to the injection of 8 · 103 lmol/kg b.w. after 30 min interval.
activation of guanylate cyclase by NO in mild HA animal Chronic HA group rats were injected daily up to 3 days
models has been reported [12] and importantly, similar with 8 · 103 lmol/kg b.w. ammonium acetate. Control
changes were also observed in these brain regions of group rats for each experimental set were simultaneously
chronic type HE patients [16]. Therefore, it is important to given with equivalent volume of physiological saline.
ascertain whether and how different brain regions respond About 80% of the rats with acute/ episodic treatment
to chronic HA with respect to O– based oxidative stress.
2 survived up to 30 min after the last injection. In case of
Brain consumes more O2 than any other tissues and thus, chronically treated rats, 90% of them could survive after
produces high level of reactive oxygen species (ROS) and the last injection. All animals were sacrificed by decapi-
operates efficient antioxidant enzyme systems to counteract tation after 30 min of the final injection and cerebral cortex
the deleterious effects of oxidative stress [17, 18]. Super- & cerebellum were dissected out, washed in ice cold saline
oxide dismutase (SOD) and catalase scavenge O– to pro-
2 (0.9% NaCl) and stored frozen at –70°C for further studies.
duce water & O2, whereas, interplay of SOD, glutathione Level of HA was ascertained by measuring ammonia
peroxidase (GPx) and glutathione reductase (GR) channels concentration in whole brain taking fresh tissues from 3
O– in a NADPH dependent pathway to maintain the ratio of
2 rats from each control as well as experimental groups.
GSH/GSSG and to prevent lipid peroxidation during
oxidative stress. It has been reported that though catalase is Preparation of tissue extracts
also found in brain cells, it is SOD-GPx-GR pathway that is
more important for antioxidant activities in brain [19, 20]. Whole brain, cerebral cortex and cerebellum extracts were
In view of a high degree of metabolic plasticity in brain prepared in 0.02 M Tris–Cl (pH 7.4) containing protease
cells in general [18] and with respect to antioxidant inhibitors as described from our lab [25]. Extracts were
enzymes in particular [21–23], it may be speculated that as centrifuged at 35,000 g for 45 min at 4°C. The superna-
compared to the acute conditions, chronic HA may produce tants collected were used for the studies on antioxidant
differential changes in the antioxidant enzyme system in enzymes and other biochemical assays. Protein content was
different brain regions. In the present report, we have determined by the method of Lowery et al. [26].
compared, in a concerted manner, the extent of oxidative
damage and levels of all the key antioxidant enzymes in rat Biochemical estimations
brain cortex (less affected due to mild HA) and cerebellum
(whose functions are affected the most in chronic HE Ammonia concentration was measured using a kit sup-
patients) in acute and chronic HA rat models. plied by Sigma–Aldrich, USA. The brain extracts were
123

3. Neurochem Res (2008) 33:103–113 105
deproteinized in 1/5 volumes of ice-cold 100 g/l trichlo- chromogen. Absorbance was measured at 560 nm using
roacetic acid, and kept on ice for 15 min. After centrifu- butanol as blank. Unit of the enzyme was defined as the
gation at 15,000 g for 15 min at 4°C, the supernatants were amount of enzyme that produced 50% inhibition of NBT
neutralized with 2.0 M KHCO3, centrifuged again and used reduction per min. and the activity was expressed as units/
for estimating ammonia. The method employed measuring mg protein.
the rate of conversion of a-ketoglutarate to glutamate cat- Catalase (EC: 1.11.1.6) was assayed following an earlier
alyzed by glutamate dehydrogenase in the presence of reported procedure [30] with some modifications. Briefly,
ammonia. The reaction mixture (1 ml) contained 50 ll of in a reaction mixture containing 0.01 M Potassium phos-
sample, 3.4 mM a-ketoglutarate and 0.23 mM reduced phate buffer (pH 7.0) and 0.1 ml of tissue extract, reaction
NADPH in 50 mM phosphate buffer (pH 7.4). The reaction was started by the addition of 0.8 M hydrogen peroxide
was started by the addition of suitably diluted glutamate (H2O2) and stopped after 60 s by 2.0 ml dichromate acetic
dehydrogenase. Initial and final (after 5 min) absorbance at acid reagent. All the tubes were heated in a boiling water
340 nm was used to calculate the concentration of bath for 10 min., cooled and absorbance was read at
ammonia in terms of lmol/g wet wt of tissue. 570 nm. After comparing with a standard plot constructed
Malondialdehyde (MDA), the product of lipid peroxi- using a range of 10–160 lmoles of H2O2, the activity of
dation, was measured by the method reported earlier [27]. catalase was expressed as lmoles of H2O2 consumed/min/
Briefly, 1 ml of Tris–Maleate buffer (0.2 M, pH 5.9) and mg protein.
0.5 ml of the extract was incubated at 37°C for 30 min.
Thereafter, 1.5 ml of thiobarbituric acid (TBA) was added Analysis of SOD and catalase by non-denaturing PAGE
and the mixture was incubated in boiling water bath for
10 min using tight condensers. After cooling, 3 ml of Non-denaturing PAGE of the tissue extracts were per-
pyridine: n-butanol mixture (3:1 v/v) and 1 ml of 1.0 N (w/ formed as reported from this laboratory [31]. For SOD, the
v) NaOH were added. The contents were thoroughly mixed extract containing 60 lg protein was loaded in each lane of
and allowed to stand for 10 min. The absorbance was read 12% non-denaturing PAGE. After electrophoresis, the gels
at 548 nm and the levels of lipid peroxidation were ex- were subjected to substrate specific staining of SOD bands
pressed as nmole MDA/g wet wt. as described earlier [32]. The staining mixture consisted of
Total thiol was estimated as described earlier [28]. 2.5 mM NBT, 28 lM riboflavin, and 28 mM TEMED.
Aliquots of 0.1 ml tissue extracts were mixed with 1.5 ml After 20 min incubation in the dark, gels were exposed to
of 0.2 M Tris buffer, pH 8.2 and 0.1 ml 0.01 M 5,5’-Di- a fluorescent light to develop achromatic bands against
thio-bis (2-nitrobenzoic acid) (DTNB) . The mixture was dark blue background corresponding to SOD protein in
made up to 10 ml with methanol and was incubated for the gel.
30 min. The mixture was then centrifuged at 3,000 rpm for For catalase, tissue extracts containing 60 lg proteins
15 min. and absorbance of the supernatant was read at were electrophoressed on 8% non-denaturing PAGE.
412 nm. The molar extinction coefficient of 13,100 was Catalase specific bands were developed according to Sun
used to calculate GSH (reduced glutathione) and values et al. [33]. Briefly, gels were soaked for 10 min in 0.003%
were presented as nmol/mg protein. H2O2 and then incubated in a staining mixture consisted of 2%
potassium ferricyanide and 2% ferric chloride. Achromatic
Studies on antioxidant enzymes catalase bands appeared against a blue–green background.
The intensity of bands was quantitated by gel densitometry
Assay of SOD and catalase using alphaimager 2200 gel documentation software.
The activity of superoxide dismutase (SOD; EC: 1.15.1.1) Active level of glutathione peroxidase
was measured following an earlier described method [29].
The reaction mixture consisted of 0.02 M sodium Glutathione peroxidase (GPx; EC:1.11.1.9) level was
pyrophosphate buffer (pH 8.3), 6.2 lM phenazine metho- determined by in gel detection method as described earlier
sulphate (PMS), 30 lM nitroblue tetrazolium (NBT), and [34]. After 10% non-denaturing PAGE of the extracts
0.1 ml suitably diluted tissue extracts. The reaction was containing 30 lg protein in each lane, the gels were
started by the addition of 50 lM NADH at 30°C and incubated in a GPx specific staining mixture composed of
stopped after 90 s by the addition of 2.0 ml glacial acetic 50 mM Tris–Cl buffer (pH 7.9), 3 mM GSH, 0.004%
acid. A control set without tissue extract was run simul- H2O2, 1.2 mM NBT and 1.6 mM PMS. Achromatic bands
taneously. The reaction mixture was stirred, shaken with corresponding to GPx activity appeared against a violet–
4 ml of n-butanol, allowed to stand for 10 min and blue background. The level of GPx was quantified by gel
centrifuged to separate butanol layer containing the densitometry as described earlier.
123

4. 106 Neurochem Res (2008) 33:103–113
During PAGE based detection of all the three antioxi- as a measure of reducing equivalents in the brain cells,
dant enzymes, SOD, catalase and GPx, development of was observed to be unaltered in both cerebral cortex and
enzyme specific bands were confirmed by comparing the cerebellum under acute and chronic HA.
results of similarly run gels stained in the presence and
absence of the enzyme specific substrates. In each case, Degree of HA & the level of antioxidant enzymes
PAGE was performed 3–4 times and mean ± SD of
densitometric values of the bands as % of control lane from In general, activity of the enzymes measured in cell free
all the gels run were presented with a representative gel extracts is correlated with the metabolic efficiency of the
photograph. cells under a variety of pathophysiological conditions.
However, measuring enzyme activity in cell free extracts
Glutathione reductase assay may not reflect actual levels of the enzymatic proteins in
the cells. Therefore, to monitor active levels of the anti-
Activity of glutathione reductase (GR; EC: 1.6.4.2) was oxidant enzymes, in the present study, cell extracts were
determined following the method of Carlberg and subjected to non-denaturing PAGE followed by activity
Mannervik [35]. In brief, the reaction mixture (1 ml) staining based detection of enzymatic proteins in the gel.
consisted of 0.2 M sodium phosphate buffer (pH 7.0), This method is relatively less sensitive than to detecting
0.2 mM EDTA, 1 mM oxidized glutathione (GSSG) and proteins by Western blotting. However, it is more relevant
0.2 mM NADPH. The reaction was initiated by the for physiological interpretations, as in this method detec-
addition of the tissue extract and oxidation of NADPH was tion is based on specificity of the enzyme for its substrate
recorded as decrease in absorbance at 340 nm for 5 min. and thus, activity based intensity of bands in gel reflects
Nonspecific oxidation of NADPH was corrected by the only active level of the enzyme (native protein). In com-
absorbance obtained in the absence of GSSG. Unit of the parison, antibody based detection can not differentiate
enzyme was defined as lmole NADP/min/ at 30°C and between the active and inactive structures of the proteins.
the enzyme activity was expressed as units/mg protein. A difference between Western blot detected enzymatic
Statistical analysis of the data was done as reported protein level and that with the intensity of activity bands in
earlier [25] and the student ‘t’ test was performed to find gel has been reported in case of most of the antioxidant
the level of significance between control and experimental enzymes in a tumor cell line [36]. Thus, in the present
groups. article, results from spectrophotometric measurements
have been interpreted as activity level of the enzyme and
PAGE bands as the level of active fraction of the enzymatic
Results protein in brain tissues.
As compared to the respective control groups, ~ 5–7 fold Effect of acute HA on antioxidant enzymes
increases in brain ammonia level was observed in rats with
episodic treatment of ammonium acetate and ~1.5–1.8 fold The first step of neutralization of O– is completed by
2
increase with those treated once daily up to 3 days. As synchronized activities of SOD & catalse and/or by SOD &
reported earlier [24], these groups were referred to as acute GPx in mammalian cells. As compared to the control group
and chronic HA groups respectively. rats, activities of SOD and catalase were observed to be
declined significantly (P < 0.01–0.001) in both, cerebral
Comparison of oxidative damage due to acute and cortex and cerebellum of rats with acute HA (Figs. 1A,
chronic HA 2A). The intensity of SOD band in gel also followed the
declining pattern in the cortex, however, with a significant
Measuring MDA level, as a stable product of lipid peroxi- (P < 0.05) increase in cerebellum of acute HA rats
dation, is a reliable tool to assess the extent of oxidative (Fig. 1B, C). And in case of catalase, intensity of PAGE
damage at cellular level. According to Table 1, as compared bands were found unchanged in both the brain regions
to the control rats, there was a significant increase (1.3 fold) during acute HA (Fig. 2B, C). Such a non-correlative
in MDA level in cerebral cortex of the rats with acute HA, pattern between the activity data and PAGE results of SOD
but with no change during chronic HA. In cerebellum, and catalase could be attributed to some inhibitory mech-
however, MDA level was 3- and 2-fold higher in acute and anisms for these enzymes in brain during acute HA.
chronic HA rats respectively than the corresponding control Four isoforms of GPx have been reported in mammalian
groups. When compared between cortex and cerebellum, tissues [20]. Though, brain contains pre-dominantly phos-
there was ~ 2 times higher MDA level in cerebellum than pholipids hydrogen peroxide GPx (pHGPx), the other three
the cortex in both the HA group rats. The level of total GSH, isoforms have also been reported in brain but in less
123

5. Neurochem Res (2008) 33:103–113 107
Table 1 Effect of acute and chronic hyperammonemia on the level of lipid peroxidation and total thiol (GSH) in cerebral cortex and cerebellum
Tissues Biochemical parameter Control Acute Control Chronic
Cerebral cortex Lipid peroxidation (MDA nmol/g wet wt) 58.65 ± 6.85 78.30 ± 5.8* 55.4 ± 5.41 55.09 ± 4.0
Total thiol (GSH) (nmol/mg protein) 1.35 ± 0.075 1.26 ± 0.125 1.37 ± 0.193 1.28 ± 0.13
Cerebellum Lipid peroxidation (MDA nmol/g wet wt) 54.50 ± 5.36 163.07 ± 7.12*** 54.72 ± 5.82 108.02 ± 8.48**
Total thiol (GSH) (nmol/mg protein) 1.28 ± 0.166 1.04 ± 0.080 1.06 ± 0.114 1.17 ± 0.075
Values are mean ± SD where n = 4 and each experiment done in duplicates
* ** ***
P < 0.05, P < 0.01, P < 0. 001 (Control versus experimental group)
amount [20]. In the absence of a literature on classification ammonia toxicity in brain, pure hyperammonemic animal
of GPx isoforms based on their migration in non-denatur- models, induced by administration of ammonium salt, with
ing PAGE, in this article, GPx bands have been referred to normal liver function is recommended over other HA
as GPx1–GPx 4 based on their relative migration in non- models with acute liver failure [37]. This is because the
denaturing PAGE starting from top to bottom (Figs. 3, 7). findings from pure HA models are assumed to be devoid of
According to Fig. 3A and B, as compared to the control the interferences from other pathological factors associated
lanes, all the four GPx isoforms though declined slightly to liver dysfunction. Additionally, ammonia diffuses in
(P < 0.05) in the cerebral cortex, but with an insignificant brain with a faster rate during HE than the normal condi-
change in cerebellum of rats with acute HA. A similar tion [38] and thus, brain ammonia level, than the concen-
pattern was also observed when GPx activity was measured tration of ammonia in blood, is considered more relevant
in vitro in the cell extracts from the respective brain re- for interpreting the data obtained using HA animal models
gions (unpublished results). Contrary to this, in comparison [24]. In the present report, we have used hyperammonemic
to the samples from control rats, though there was a small rats induced by administration of ammonium acetate
decline (P < 0.05) in the activity of GR in the cerebellum, wherein, as reported earlier [24], ~5–7 and 1.5–1.8 fold
GR activity in the cerebral cortex remained unchanged increases in brain ammonia level was considered as acute
during acute HA. and chronic HA groups respectively.
Brain processes ~20% of O2 consumed by the whole
Effect of chronic HA on antioxidant enzymes body for generating ATP via oxidative phosphorylation in
mitochondria and therefore, brain cells are consistently
Figures 5–7 illustrate that in cerebral cortex of chronic HA exposed to high ROS. Abundance of myelinated nerve
rats, as compared to the control group, activities as well as fibers makes brain enriched with phospholipids containing
levels of active fractions of SOD, catalase and all isoforms poly unsaturated fatty acids, and thus, brain cells become
of GPx increased significantly (P < 0.05–0.001). However, highly prone to ROS dependent derangements in mem-
in cerebellum, though the activity and active levels of SOD brane structure and functions [39]. The level of lipid
(P < 0.001) & all the GPx bands including GPx 2 (pHGPx) peroxidation is a good indicator to assess the extent of
declined significantly (P < 0.05), there were no significant oxidative damage produced by ROS in the brain. The over
change observed in the activity and the level of catalase activation of NMDA receptors [1] and ammonia induced
during chronic HA. Moreover, as compared to the control mitochondrial dysfunction [4, 5] could be the main source
group rats, rats with chronic HA showed significant decline of excess of ROS in brain during HA. The rate of free
(P < 0.01) in the activity of GR in cerebral cortex but with radical production and the level of lipid peroxidation have
no change in cerebellum (Fig. 8). been reported to be significantly high in the whole brain of
acute HE rats [40]. According to Table 1, however, when
compared between the cerebral cortex and the cerebellum
Discussion in pure HA rats, significantly increased level of lipid per-
oxidation (~ 2 times higher) in cerebellum than the cortex
In the present article, we intended to address two aspects of under both acute and chronic conditions clearly suggest
ammonia neurotoxicity, one the relationship between the that cerebellum is more susceptible for oxidative damage
degree of HA and oxidative stress in brain & secondly, due to ammonia toxicity than the cortex. Furthermore, ~ 3
since cerebellum associated functions are affected the most and 2 fold increases in the MDA level in cerebellum of
during chronic HA, is it that cerebellum is more susceptible acute and chronic HA rats respectively suggest for a cause
to ammonia toxicity than other brain regions with respect and effect relationship between the degree of HA in brain
to oxidative stress. For such comparative studies on and the oxidative damage in cerebellum. Nonetheless,
123

6. 108 Neurochem Res (2008) 33:103–113
A control
A C ontrol
12 4
HA
Catalase (U/mg protein)
HA 3.5
10
3
SOD (U/mg protein)
** 2.5
8
2
6 1.5 ***
***
1 ***
4
0.5
2 0
Cerebral c ortex Cerebellum
0
Cerebral cortex Cerebellum B C o n t ro l HA Control HA
B C o nt r ol HA Control HA Catalase
SOD
C 120
100
C
% of control
140 80
Control
*
120 60
HA
100 40
% of control
80 ** 20
60 0
40 Cerebral c ortex Cerebellum
20 Fig. 2 Effect of acute hyperammonemia on activity (A) and level of
active catalase protein (B & C) in cerebral cortex and cerebellum of
0
Cerebral cortex Cerebellum
rats. The values in A represent mean ± SD where n = 4 and each
experiment done in duplicates. In case of B, pooled tissue extracts
Fig. 1 Effect of acute hyperammonemia on activity (A) and level of from 4 rats containing 60 lg protein in each lane was electropho-
active SOD protein (B & C) in cerebral cortex and cerebellum of rats. ressed on 8% non- denaturing PAGE followed by substrate specific
The values in A represent mean ± SD where n = 4 and each development of catalase bands. The gel photograph in B is a
experiment done in duplicate. In case of B, pooled tissue extracts representative out of the 3 PAGE repeats. In panel C, relative
from 4 rats containing 60 lg protein in each lane was electropho- densitometric values of catalase bands from experimental group as %
ressed on 12% non- denaturing PAGE followed by substrate specific of the control lane have been presented as mean ± SD from the 3
development of SOD bands. The gel photograph in B is a PAGE repeat experiments. ***P < 0.001 (control versus experimental
representative out of the 4 PAGE repeats. In panel C, relative groups)
densitometric values of SOD bands from experimental group as % of
the control lane have been presented as mean ± SD from the 4 PAGE
repeat experiments *P < 0.05, **P < 0.01, ***P < 0.001 (control
brain regions under acute as well as chronic HA conditions
versus experimental groups)
(Table 1). In the cellular antioxidant pathway, the turnover
of GSH/GSSG is regulated by synchronized activities of
cortex showed resistance to HA dependent oxidative stress, GPx and GR in mammalian cells. Both these enzymes did
as there was no change in MDA level in the cortex of not show much alternation, except a moderate decrease in
chronic but with a mild (1.3 fold) increase in that from GPx and GR in cortex and cerebellum respectively
acute HA rats. (Figs. 3, 4), due to acute HA, and thus, could be correlated
The level of reduced glutathione (GSH), a tripeptide with the unchanged level of GSH in both the brain regions
responsible to maintain reducing equivalents under oxida- during acute HA. However, significantly opposite trends of
tive stress, is another critical factor to assess the level of GPx and GR in the cerebral cortex of chronic HA rats
oxidative stress in mammalian cells. Interestingly, there (Figs. 7, 8) did not correlate with the unchanged level of
was no significant change in the level of GSH in both the GSH in the cortex of rats with chronic HA. It is suggested
123

7. Neurochem Res (2008) 33:103–113 109
A 40
Control HA Control HA Control
35
GPx 1 HA
30
GR (U/mg protein)
GPx 2 25
GPx 3 *
20
GPx 4
15
10
B
140 5
120 0
Cerebral cortex Cerebellum
100
% of control
*
Fig. 4 Effect of acute hyperammonemia on activity of GR in cerebral
80
cortex and cerebellum of rats. The values represent mean ± SD where
60 n = 4 and each experiment done in duplicates. *P < 0.05 (control
versus experimental groups)
40
20
mitochondria, is converted to H2O2 by SOD. Simultaneous
0
removal of H2O2 by either catalse and/or by GPx is crucial
Cerebral c ortex Cerebellum for preventing membrane damage due to oxidative stress.
Fig. 3 Effect of acute hyperammonemia on level of active GPx In brain, SOD-GPx-GR pathway is considered to play
protein in cerebral cortex and cerebellum of rats. In case of A, pooled major role of antioxidant activities [19, 20]. With the
tissue extracts from 4 rats containing 30 lg protein in each lane was increased production of ROS, most of these enzymes were
electrophoressed on 10% non-denaturing PAGE followed by substrate found to be declined in whole brain of rat with acute HE
specific development of GPx bands. The gel photograph in A is a
representative out of the 4 PAGE repeats. In panel B, relative [8]. However, according to the results presented here, when
densitometric values of GPx bands from experimental group as % of the levels of all these enzymes were compared in concerted
the control lane have been presented as mean ± SD from the 4 PAGE manner in two different brain regions (cerebral cortex and
repeat experiments. *P < 0.05 (control versus experimental group) cerebellum) under acute and chronic HA, changes in all
these enzymes were found to differ as a function of degree
that a highly adaptive metabolic coupling operates between of HA but with a regional specificity. In cerebellum,
astrocytes and neurons to maintain the normal level of this though GPx showed resistance against acute HA, there was
tripeptide under unphysiological conditions in brain [41, a significant decline in the levels of SOD, catalase and GR
42]. When neuron’s GSH gets depleted due to acute under acute HA and thus, suggested for acute HA depen-
ammonia intoxication, the precursors for GSH synthesis dent oxidative stress in rat cerebellum. It was also corre-
are supplied from astrocytes which are supposed to be less lated well with a significant increase in the level of lipid
susceptible to ROS insult [41]. Furthermore, gamma glut- peroxidation in cerebellum of acute HA rats (Table 1).
amyl-cystein synthetase is also responsible to produce GSH Cerebral cortex also showed significant decline in SOD and
in the cells, and this enzyme has been reported to be in- catalase, however, with a moderate decrease in GPx and no
creased in the astrocytes under acute HA condition [43]. change in the level of GR under acute HA (Figs. 3, 4). In
Thus, it is likely that these additional routes could con- view of relatively less increase in the level of lipid
tribute for maintaining GSH level in the cortex of chronic peroxidation due to acute HA in the cortex (~ 2 times less
HA rats even when GR activity declined significantly than cerebellum), it may be assumed that resistance of GPx
(P < 0.01). Similar argument may be given for the unal- and GR to acute HA might be accountable to prevent
tered level of GSH in cerebellum of chronic HA rats where, oxidative damage in cerebral cortex even at the face of
GPx showed significant decline (P < 0.05) but with a little significant decline in SOD and catalase.
change in GR activity (Figs. 7, 8). During chronic HA, a significant decline in the level of
The changes in the levels of antioxidant enzymes during SOD (both by activity and PAGE results) and GPx (Figs. 5,
oxidative stress are the most critical factors in determining 7) coincided with the significant increase in the level of
the extent of oxidative damage produced by ROS during lipid peroxidation in cerebellum (Table 1), however, with
neuropathology [17, 44]. All parts of brain contain SOD, no change in the level of catalase and GR (Figs. 6, 8).
catalase, GPx and GR in high concentration to counter This suggests that decline in the level of SOD and GPx
balance the deleterious effects of ROS [44, 45]. Excess are mainly accountable to allow oxidative damage in
of superoxide anion (O–), the major ROS produced in
2
cerebellum and unaffected GR plays a permissive role in
123

8. 110 Neurochem Res (2008) 33:103–113
A 16 A 7
**
Control *** Control
14 6
Catalase (U/mg protein)
SOD (U/mg protein)
HA HA
12 5
10
4
8
*** 3
6
4 2
2 1
0 0
Cerebral c ortex Cerebellum
Cerebral cortex Cerebellum
B Control HA Control HA
B C o n t ro l HA Control HA
Catalase
SOD
C 140
*
C 120
180
100
% of control
160 **
80
140
60
% of control
120
100 40
80 ** 20
60 0
40 Cerebral cortex Cerebellum
20
0 Fig. 6 Effect of chronic hyperammonemia on activity (A) and level
Cerebral c ortex Cerebellum of active catalase protein (B & C) in cerebral cortex and cerebellum
of rats. The values in A represent mean ± SD where n = 4 and each
Fig. 5 Effect of chronic hyperammonemia on activity (A) and level experiment done in duplicates. In case of B, pooled tissue extracts
of active SOD protein (B & C) in cerebral cortex and cerebellum of from 4 rats containing 60 lg protein in each lane was electropho-
rats. The values in A represent mean ± SD where n = 4 and each ressed on 8% non-denaturing PAGE followed by substrate specific
experiment done in duplicate. In case of B, pooled tissue extracts development of catalase bands. The gel photograph in B is a
from 4 rats containing 60 lg protein in each lane was electropho- representative out of the 3 PAGE repeats. In panel C, relative
ressed on 12% non-denaturing PAGE followed by substrate specific densitometric values of catalase bands from experimental group as %
development of SOD bands. The gel photograph in B is a of the control lane have been presented as mean ± SD from the 3
representative out of the 4 PAGE repeats. In panel C, relative PAGE repeat experiments. *P < 0.05, ***P < 0.001 (control versus
densitometric values of SOD bands from experimental group as % of experimental groups)
the control lane have been presented as mean ± SD from the 4 PAGE
repeat experiments. **P < 0.01, ***P < 0.001 (control versus exper-
imental groups) [18]. The whole brain of rats pre-exposed to chronic HA
have been found to resist the changes in the level of crucial
maintaining the normal level of GSH (Table 1) during metabolites which are normally produced otherwise during
chronic HA in this brain region. This again supports the acute HA [8]. At the face of significant decline in the
view that SOD and GPx are the most critical antioxidant activity of most of the antioxidant enzymes, SOD activity
enzymes in brain [19, 20]. Nonetheless, since, both these was reported to be increased significantly in all the brain
enzymes declined specifically in cerebellum (as compared regions of rats with fulminate liver type acute HE [10]. As
to the cortex) and that cerebellum associated functions are per the results presented here, however, it is evident that
affected the most in HE patients [14, 15], it may be argued chronic HA produces adaptive changes only in cerebral
that relatively greater susceptibility of cerebellum for cortex with respect to the SOD-GPx pathway in particular.
ammonia toxicity dependent antioxidant defense could be This could contribute for relatively less effect of chronic
accountable for pathogenesis of low grade chronic HA. HA on the cortex associated function than the cerebellum
In case of cortex, contrary to the effect of acute (Fig. 8).
ammonia exposure, chronic HA produced significant in- It has been suggested that each antioxidant enzyme
creases in the levels of SOD, catalase and GPx (Figs. 5–7) has a functionally distinct role, or cooperates with other
and thus, suggested positive adaptation in brain cortex enzymes to protect the cell under a variety of pathophysi-
against a low grade chronic HA with respect to these ological conditions [46] and thus, HA dependent differen-
antioxidant enzymes. Brain is considered to be a highly tial changes in the set of antioxidant enzymes e.g. up
plastic tissue so far metabolic adaptations are concerned regulation of SOD-GPx in cortex and their down regulation
123

9. Neurochem Res (2008) 33:103–113 111
A 35
Control HA Control HA Control
30 HA
GPx 1
GR (U/mg protein)
25
**
20
GPx 2
15
GPx 3
GPx 4 10
5
B 140.00 0
Cerebral cortex Cerebellum
120.00 *
100.00 Fig. 8 Effect of chronic hyperammonemia on activity of GR in
% of control
cerebral cortex and cerebellum of rats. The values represent
80.00 * mean ± SD where n = 4 and each experiment done in duplicates.
**
P < 0.01 (control versus experimental groups)
60.00
40.00
resulted due to SOD and catalase inhibitory conditions
20.00 induced in brain during acute HA. H2O2 is a known
0.00 physiological inhibitor of SOD [47, 48] and has been
Cerebral c ortex Cerebellum demonstrated recently to inhibit specific isoforms of this
enzyme in brain [49]. Increased accumulation of Mn2+ in
Fig. 7 Effect of chronic hyperammonemia on level of active GPx brain is associated with Alzheimers type II astrocytosis
protein in cerebral cortex and cerebellum of rats. In case of A, pooled
tissue extracts from 4 rats containing 30 lg protein in each lane was [50], a hall mark of acute HA [1] and as reviewed by
electrophoressed on 10% non-denaturing PAGE followed by substrate Takeda [51], increased level of Mn2+ inhibits catalase and
specific development of GPx bands. The gel photograph in A is a also induces a burst of H2O2 in brain cells. Also, as per the
representative out of the 4 PAGE repeats. In panel B, relative results presented here (Fig. 2A), a drastic decrease in the
densitometric values of GPx bands from experimental group as % of
the control lane have been presented as mean ± SD from the 4 PAGE activity of catalase in cerebellum of acute HA rats may also
repeat experiments. *P < 0.05 (control versus experimental group) contribute for an unusual increase in H2O2 and thus, can
further potentiate inhibition of SOD in this brain region
during acute HA. A two times higher level of MDA in
in cerebellum during chronic HA could be the result of cerebellum than the cortex of acute HA rats (Table 1)
differential sensitivity of cortex and cerebellum to chronic provide additional support to this argument. Furthermore, it
HA. Opposite responses of cortex and cerebellum to NO has been demonstrated that inhibition of SOD at cellular
dependent signaling pathway during HA in rats [12] and level induces increase in the mRNA level of this enzyme
also in HE patients [16] provide support to this argument. [52], and SOD proteins are highly resistant to denaturation
Such a pattern has been shown in other neurological & oxidative damage even at a high concentration of H2O2
disorders also. Different antioxidant enzymes showed [48]. Therefore, it is likely that inactivation of SOD
differential alterations in the brain of patients with observed in cell free extracts due to increased oxidative
Alzheimers type dementia [21] and also in D-amphitamine burst in cerebellum of acute HA rats might not be reflected
induced neurotoxicity [22]. Increase in the level of SOD at protein level (Fig. 1). Accordingly, since inactivation
and catalase in different brain regions of rats with mala- of SOD would be expected to be minimal during mild
thion-induced oxidative stress is another example of oxidative stress, in vitro activity data and level of SOD
adaptive changes in antioxidant enzymes [23]. protein should be mutually correlative. And indeed, a
With a view to have a molecular rationale behind similar pattern of SOD profile was observed in the cerebral
significant changes in the activities of SOD, catalase and cortex of acute HA rats (Fig. 1) with ~ 2 times less
GPx during HA, these enzymes were further analyzed on oxidative stress than cerebellum (Table 1, MDA data). As
PAGE. It was interesting to note that while level of SOD Mn2+ also inhibits catalase in the brain [51] and such
protein increased in cerebellum of rats with acute HA transitory metal-protein interaction is likely to get disso-
(Figs. 1B, C), activity of this enzyme (when measured ciated during electrophoresis, a similar argument may be
in vitro) showed significant decline (Fig. 1A). Similar given for significant decreases in the activity of catalase
pattern was observed with catalase in both the brain regions in vitro but with insignificant change in its level on PAGE
of acute HA rats (Fig. 2A–C). Such a mismatch could be analysis in both the brain regions of acute HA rats. These
123

10. 112 Neurochem Res (2008) 33:103–113
arguments get further support from a uniform correlative 11. Hermenegildo C, Montoliu C, Llansola M et al (1998) Chronic
pattern observed between in vitro data and PAGE patterns hyperammonemia impairs the glutamate-nitric oxide-cyclicGMP
pathway in cerebellar neurons in culture and in the rat in vivo.
of SOD and catalase in both the brain regions of chronic Eur J Neurosci 10:3201–3209
HA rats (Figs. 5, 6) showing significantly less oxidative 12. Rodrigo R, Felipo V (2006) Brain regional alternations in the
stress as compared to the acute HA rats (Table 1, MDA modulation of the glutamate-nitric oxide-cGMP pathway in liver
data). Thus, it is evident that the extent of oxidative stress cirrhosis: role of hyperammonemia and cell types involved.
Neorochem Int 48:472–477
induced during acute HA acts as an additional factor in 13. Kosenko E, Kaminsky YG, Felipo V et al (1993) Chronic
modulating the activities of SOD and catalase irrespective hyperammonemia prevents changes in brain energy and ammonia
of the actual levels of these proteins in both the brain metabolism induced by acute ammonia intoxication. Biochim
regions. Biophys Acta 1180:321–326
14. Gilberstadt SJ, Gilberstadt H, Zieve L et al (1980) Psychomotor
In conclusion, active levels of all the antioxidant enzymes performance defects in cirrhotic patients without overt encepha-
were found altered differently in cerebral cortex and cere- lopathy. Arch Int Med 140:519–521
bellum as a function of degree of HA. As compared to a 15. Tarter RE, Hegedus AM, Van Thiel DH et al (1984) Non-
uniform decline in the activities of most of the antioxidant alcoholic cirrhosis associated with neuropsychological dysfunc-
tion in the absence of overt evidence of hepatic encephalopathy.
enzymes due to acute HA, chronic HA was found to induce Gastroenterology 86:1421–1427
brain region specific changes which are likely to render 16. Corbalan R, Chaturet N, Behrends S et al (2002) Region selective
cerebellum susceptible and cerebral cortex resistant to the alternation of soluble guanylate cyclase content and modulation
oxidative stress during chronic HA. Since, cerebellum asso- in brain of cirrhotic patients. Hepatology 6:1155–1162
17. Mates JM (2000) Effects of antioxidant enzymes in the molecular
ciated functions are mainly affected during low grade chronic control of reactive oxygen species toxicology. Toxicology
HA, such changes in antioxidant enzymes might be impli- 153:83–104
cated in the encephalopathy of chronic HA. 18. Magistretti PJ (2003) Brain energy metabolism. In: Fundamental
neuroscience, 2nd edn. Elsevier Science, New York, pp339–360
Acknowledgments This work was financially supported by a DAE: 19. Tureen JF (2003) Mitochondrial formation of reactive oxygen
BRNS grant (P-29/64) to SKT. The instrumental facilities provided species. J Physiol 552:335–344
by the DST FIST and UGC-CAS Program to the department of 20. Flohe RB (1999) Tissue-specific function of individual glutathi-
Zoology are also acknowledged. one peroxidases. Free Radical Biol Med 27:951–965
21. Gsell W, Conrad R, Hickethier M et al (1995) Decreased catalase
activity but unchanged superoxide dismutase activity in brain of
patients of Alzheimer type. J Neurochem 64:1216–1233
References 22. Frey BN, Valvassori SS, Reus GS et al (2006) Changes in antioxidant
defense enzymes after D-amphetamine exposure: implications as
1. Felipo V, Butterworth RF (2002) Neurobiology of ammonia. an animal model of mania. Neurochem Res 31:699–703
Prog Neurobiol 67:259–279 23. Furtunato JJ, Feier G, Vitali AM (2006) Malathione-induced
2. Butterworth RF (2000) Hepatic encephalopathy: a neuropsychi- oxidative stress in rat brain regions. Neurochem Res 31:671–678
atric disorder involving multiple neurotransmitter systems. Curr 24. Hilgier W, Albrecht J, Lisy V et al (1990) The effect of acute and
Opin Neurol 13:721–727 chronic hyperammonemia on y-glutamyl transpeptidase in vari-
3. Marcaida G, Felipo V, Hermengildo C et al (1992) Acute ous brain regions of young and adult rats. Mol Chem Neuropathol
ammonia toxicity is mediated by the NMDA type of glutamate 13:47–56
receptor. FEBS Lett 296:67–68 25. Pandey P, Singh SK, Trigun SK (2005) Developing brain of
4. Rama Rao KV, Norenberg MD (2001) Cerebral energy metabo- moderately hypothyroid mice shows adaptive changes in the key
lism in hepatic encephalopathy and hyperammonemia. Metab enzymes of glucose metabolism. Neurol Psych Brain Res 12:159–164
Brain Dis 16:67–78 26. Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein mea-
5. Kosenko E, Felipo V, Montoliu C et al (1996) Effects of acute surement with the Folin phenol reagent. J Biol Chem 193:
hyperammonemia in vivo on oxidative metabolism in nonsy- 265–275
naptic rat brain mitochondria. Metab Brain Dis 12:69–82 27. Placer ZA, Cushman LL, Johnson BC (1966) Estimation of
6. Rama Rao KV, Jayakumar AR, Norenberg MD (2005) Role of product of lipid peroxidation (malonyl dialdehyde) in biochemi-
oxidative stress in the ammonia-induced mitochondrial perme- cal systems. Anal Biochem 16: 359–364
ability transition in cultered astrocytes. Neurochem Int 47:31–38 28. Sedlak J, Raymond HL (1968) Estimation of total thiol, protein
7. Murthy CRK, Rama Rao KV, Bai G et al (2001) Ammonia in- bound, and non protein sulfhydryl groups in tissue with Ellman’s
duced production of free radicals in primary culture of rat as- reagent. Anal Biochem 25:192–205
trocytes. J Neurosci Res 66:282–288 29. Kakkar P, Das B, Viswanathan PN (1984) A modified spectro-
8. Kosenko E, Kaminski Y, Lopata O et al (1999) Blocking of photometric assay of superoxide dismutase ( SOD). Ind J
NMDA receptors prevents the oxidative stress induced by acute Biochem Biophys 21:130–132
ammonia intoxication. Free Radical Res 26:1369–1374 30. Sinha KA (1972) Colorimetric assay of catalase. Anal Biochem
9. Norenberg MD, Jayakumar AR, Rama Rao KV (2004) Oxidative 47:389–394
stress in the pathogenesis of hepatic encephalopathy. Metab Brain 31. Trigun SK, Singh AP, Asthana RK et al (2006) Assessment of
Dis 19:313–329 bioactivity of a Fischerella species colonizing Azadirachta Indica
10. Sathyasaikumar KV, Swapna I, Reddy PVB et al (2007) (neem) Bark. Appl Ecol Environ Res 4:119–128
Fulminant hepatic failure in rats induces oxidative stress differ- 32. Beauchamp C, Fridovich I (1971) Superoxide dismu-
entially in cerebral cortex, cerebellum and pons medula. Neuro- tase:improved assays and an assay applicable to acrylamide gels.
chem Res 32:517–524 Anal Biochem 44:276–287
123

11. Neurochem Res (2008) 33:103–113 113
33. Sun Y, Elwell JH, Oberley LW (1988) A simultaneous visuali- 43. Murthy Ch RK, Bender AS, Dombro RS et al (2000) Elevation of
zation of the antioxidant enzymes glutathione peroxidase and glutathione level by ammonium ions in primary cultures of rat
catalase on polyacrylamide gels. Free Radical Res Commun astrocytes. Neurochem Int 37:255–268
5:67–75 44. Halliwell B (2001) Role of free radicals in the neurodegenerative
34. Lin CL, Chen HJ, Hou WC (2002) Activity staining of gluta- diseases: therapeutic implications for antioxidant treatment. Drug
thione peroxidase after electrophoresis on native and sodium Aging 18:685–716
dodecylsulfate polyacrylamide gels. Electrophoresis 23:513–516 45. Patenaude A, Murthy MR, Mirault ME (2005) Emerging roles of
35. Carlberg I, Mannervik B (1975) Purification and characterization thioredoxin cycle enzymes in the central nervous system. Cell
of the flavoenzyme glutathione reductase from rat liver. J Biol Mol Life Sci 62:1063–1080
Chem 250:5475–5480 46. Imai H, Nakagawa Y (2003) Biological significance of phos-
36. Wang H (2000) Over expression of L-PhGPx in MCF-7 cells. pholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4)
In: The role of mitochondrial phospholipids hydroperoxide glu- in mammalian cells. Free Radical Biol Med 34:145–169
tathione peroxide in cancer therapy. Ph.D. thesis. The University 47. Bray RC, Cockel SA, Fielden EM et al (1974) Reduction and
of Iowa, Iowa, pp16–56 inactivation of superoxide dismutase by hydrogen peroxide.
37. Kanamori K, Ross BD, Chung JC et al (1996) Severity of hy- Biochem J 139:43–48
perammonemic encephalopathy correlates with brain ammonia 48. Salo DC, Pacifici RE, Lin SW (1990) Superoxide dismutase
level and saturation of glutmine synthetase in vivo. J Neurochem undergoes proteolysis and fragmentation following oxidative
67:1584–1594 modification and inactivation. J Biol Chem 265:11919–11927
38. Lockwood AH (2004) Blood ammonia levels and hepatic 49. Mokni M, Elkahoui S, Limam F et al (2007) Effect of resveratrol
encephalopathy. Metab Brain Dis 19:345–349 on antioxidant enzyme activities in the brain of healthy rat.
39. Halliwell B, Gutteridge JMC (1985) Oxygen radicals and nervous Neurochem Res 32:981–987
system. Trends Neurosci 8:22–26 50. Hazell AS, Normandin L, Norenberg MD et al (2006) Alzheimer
40. Kosenko E, Venediktova N, Kamanisky Y et al (2003) Sources of type II astrocytic changes following sub-acute exposure to
oxygen radical in brain in acute ammonia intoxication in vivo. manganese and its prevention by antioxidant treatment. Neurosci
Brain Res 981:193–200 Lett 396:167–171
41. Dringen R, Pawlowski PG, Hirrlinger J (2005) Peroxide detoxi- 51. Takeda A (2003) Manganese action in brain function. Brain Res
fication by brain cells. J Neurosci Res 79:157–165 Rev 41:79–87
42. Hovatta I, Tennant RS, Helton R et al (2005) Glyoxalase 1 and 52. Maitre B, Jornot L, Jonod AF (1993) Effects of inhibition of
glutathione reductase 1 regulate anxiety in mice. Nature 438: catalase and superoxide dismutase activity on antioxidant enzyme
662–666 mRNA levels. Am J Physiol 265:636–643
123