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Acute and chronic hyperammonemia modulate antioxidant


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Acute and chronic hyperammonemia modulate antioxidant

  1. 1. Neurochem Res (2008) 33:103–113DOI 10.1007/s11064-007-9422-x ORIGINAL PAPERAcute and Chronic Hyperammonemia Modulate AntioxidantEnzymes Differently in Cerebral Cortex and CerebellumSantosh Singh Æ Raj K. Koiri Æ Surendra Kumar TrigunAccepted: 18 June 2007 / Published online: 4 August 2007Ó Springer Science+Business Media, LLC 2007Abstract Studies on acute hyperammonemic models stress. This is supported by ~ 2- and 3-times increases in thesuggest a role of oxidative stress in neuropathology of level of lipid peroxidation in cerebellum during chronic andammonia toxicity. Mostly, a low grade chronic type acute HA respectively, however, with no change in thehyperammonemia (HA) prevails in patients with liver cortex due to chronic HA.diseases and causes derangements mainly in cerebellumassociated functions. To understand whether cerebellum Keywords Hyperammonemia Á Ammonia neurotoxicity Áresponds differently than other brain regions to chronic type Antioxidant enzymes Á Oxidative stress Á Cerebral cortex ÁHA with respect to oxidative stress, this article compares Cerebellumactive levels of all the antioxidant enzymes vis a vis extentof oxidative damage in cerebral cortex and cerebellum ofrats with acute and chronic HA induced by intra-peritoneal Introductioninjection of ammonium acetate (successive doses of10 · 103 & 8 · 103 lmol/kg b.w. at 30 min interval for Hepatic encephalopathy (HE) is a serious nervous systemacute and 8 · 103 lmol/kg b.w. daily up to 3 days for disorder developed due to increased ammonia level in brainchronic HA). As compared to the respective control sets, resulting from liver dysfunction. This is of great concerncerebral 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 reportedacute HA rats, SOD, catalase and GR though declined that acute ammonia exposure of brain cells causessignificantly, GPx level was found to be stable. Contrary to dysfunction of multiple neurotransmitter system [1, 2] andthis, during chronic HA, levels of SOD, catalase and GPx glutamate & ammonia mediated excitotoxicity of neuronsincreased 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 stressbellum. 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 aHowever, chronic HA induces adaptive changes, with role of oxidative stress in ammonia neurotoxicity haverespect to the critical antioxidant enzymes, in cerebral cor- been derived either from cell culture studies [6, 7] and/ortex 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 longS. Singh Á R. K. Koiri Á S. K. Trigun (&) term drug abuse. Therefore, it is important to understandBiochemistry & Molecular Biology Laboratory, Department ofZoology, Banaras Hindu University, Varanasi 221005, India how chronic HA affects cellular antioxidant defensee-mail:; mechanisms in susceptible brain regions. 123
  2. 2. 104 Neurochem Res (2008) 33:103–113 There are some reports on the role of oxidative stress in Experimental procedurechronic hyperammonemic models also; however, most ofthem are focused to the hyperammonemia (HA) dependent Animal and chemicalsimpairment of NMDA receptor activity [1] via alterationsin 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 fromin brain energy and ammonia metabolites, which are institutional ethical committee for the care and use ofaltered 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 bestchondrial 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, Coomassieoxygen 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 fluorideof 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 designdisturbances, expressionless face, rigid muscle tone, tremoretc, 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], forThus, it is likely that cerebellum responds differently to acute HA, first 10 · 103 lmol/kg b.w. of ammoniumHA than the other brain regions. Differential susceptibility acetate was administered to the rats followed by a secondof 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 daysmodels has been reported [12] and importantly, similar with 8 · 103 lmol/kg b.w. ammonium acetate. Controlchanges were also observed in these brain regions of group rats for each experimental set were simultaneouslychronic 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 treatmentto 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 afterproduces 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 cortexthe deleterious effects of oxidative stress [17, 18]. Super- & cerebellum were dissected out, washed in ice cold salineoxide 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 ammoniaperoxidase (GPx) and glutathione reductase (GR) channels concentration in whole brain taking fresh tissues from 3O– 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 duringoxidative stress. It has been reported that though catalase is Preparation of tissue extractsalso found in brain cells, it is SOD-GPx-GR pathway that ismore important for antioxidant activities in brain [19, 20]. Whole brain, cerebral cortex and cerebellum extracts wereIn view of a high degree of metabolic plasticity in brain prepared in 0.02 M Tris–Cl (pH 7.4) containing proteasecells in general [18] and with respect to antioxidant inhibitors as described from our lab [25]. Extracts wereenzymes 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 antioxidantdifferential changes in the antioxidant enzyme system in enzymes and other biochemical assays. Protein content wasdifferent 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 oxidativedamage and levels of all the key antioxidant enzymes in rat Biochemical estimationsbrain 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 were123
  3. 3. Neurochem Res (2008) 33:103–113 105deproteinized in 1/5 volumes of ice-cold 100 g/l trichlo- chromogen. Absorbance was measured at 560 nm usingroacetic acid, and kept on ice for 15 min. After centrifu- butanol as blank. Unit of the enzyme was defined as thegation at 15,000 g for 15 min at 4°C, the supernatants were amount of enzyme that produced 50% inhibition of NBTneutralized 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: was assayed following an earlieralyzed 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, reactionNADPH in 50 mM phosphate buffer (pH 7.4). The reaction was started by the addition of 0.8 M hydrogen peroxidewas started by the addition of suitably diluted glutamate (H2O2) and stopped after 60 s by 2.0 ml dichromate aceticdehydrogenase. Initial and final (after 5 min) absorbance at acid reagent. All the tubes were heated in a boiling water340 nm was used to calculate the concentration of bath for 10 min., cooled and absorbance was read atammonia 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 ofdation, 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 PAGEand the mixture was incubated in boiling water bath for10 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, thev) NaOH were added. The contents were thoroughly mixed extract containing 60 lg protein was loaded in each lane ofand allowed to stand for 10 min. The absorbance was read 12% non-denaturing PAGE. After electrophoresis, the gelsat 548 nm and the levels of lipid peroxidation were ex- were subjected to substrate specific staining of SOD bandspressed 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 toof 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 againstthio-bis (2-nitrobenzoic acid) (DTNB) . The mixture was dark blue background corresponding to SOD protein inmade 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 proteins15 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 Sunused 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. AchromaticStudies on antioxidant enzymes catalase bands appeared against a blue–green background. The intensity of bands was quantitated by gel densitometryAssay of SOD and catalase using alphaimager 2200 gel documentation software.The activity of superoxide dismutase (SOD; EC: Active level of glutathione peroxidasewas measured following an earlier described method [29].The reaction mixture consisted of 0.02 M sodium Glutathione peroxidase (GPx; EC: level waspyrophosphate buffer (pH 8.3), 6.2 lM phenazine metho- determined by in gel detection method as described earliersulphate (PMS), 30 lM nitroblue tetrazolium (NBT), and [34]. After 10% non-denaturing PAGE of the extracts0.1 ml suitably diluted tissue extracts. The reaction was containing 30 lg protein in each lane, the gels werestarted by the addition of 50 lM NADH at 30°C and incubated in a GPx specific staining mixture composed ofstopped 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 bandstaneously. 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 gelcentrifuged to separate butanol layer containing the densitometry as described earlier. 123
  4. 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 andenzyme specific bands were confirmed by comparing the cerebellum under acute and chronic HA.results of similarly run gels stained in the presence andabsence of the enzyme specific substrates. In each case, Degree of HA & the level of antioxidant enzymesPAGE was performed 3–4 times and mean ± SD ofdensitometric values of the bands as % of control lane from In general, activity of the enzymes measured in cell freeall the gels run were presented with a representative gel extracts is correlated with the metabolic efficiency of thephotograph. cells under a variety of pathophysiological conditions. However, measuring enzyme activity in cell free extractsGlutathione 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: was oxidant enzymes, in the present study, cell extracts weredetermined following the method of Carlberg and subjected to non-denaturing PAGE followed by activityMannervik [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 detecting0.2 mM EDTA, 1 mM oxidized glutathione (GSSG) and proteins by Western blotting. However, it is more relevant0.2 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 substraterecorded as decrease in absorbance at 340 nm for 5 min. and thus, activity based intensity of bands in gel reflectsNonspecific 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 differentiateenzyme 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 inearlier [25] and the student ‘t’ test was performed to find gel has been reported in case of most of the antioxidantthe level of significance between control and experimental enzymes in a tumor cell line [36]. Thus, in the presentgroups. 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 enzymaticResults protein in brain tissues.As compared to the respective control groups, ~ 5–7 fold Effect of acute HA on antioxidant enzymesincreases in brain ammonia level was observed in rats withepisodic treatment of ammonium acetate and ~1.5–1.8 fold The first step of neutralization of O– is completed by 2increase 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 groupand chronic HA groups respectively. rats, activities of SOD and catalase were observed to be declined significantly (P < 0.01–0.001) in both, cerebralComparison 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 significantMeasuring MDA level, as a stable product of lipid peroxi- (P < 0.05) increase in cerebellum of acute HA ratsdation, is a reliable tool to assess the extent of oxidative (Fig. 1B, C). And in case of catalase, intensity of PAGEdamage at cellular level. According to Table 1, as compared bands were found unchanged in both the brain regionsto the control rats, there was a significant increase (1.3 fold) during acute HA (Fig. 2B, C). Such a non-correlativein MDA level in cerebral cortex of the rats with acute HA, pattern between the activity data and PAGE results of SODbut 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 mammaliangroups. 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 threethe cortex in both the HA group rats. The level of total GSH, isoforms have also been reported in brain but in less123
  5. 5. Neurochem Res (2008) 33:103–113 107Table 1 Effect of acute and chronic hyperammonemia on the level of lipid peroxidation and total thiol (GSH) in cerebral cortex and cerebellumTissues Biochemical parameter Control Acute Control ChronicCerebral 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 ± 0.13Cerebellum 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 ± 0.075Values 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 animalof GPx isoforms based on their migration in non-denatur- models, induced by administration of ammonium salt, withing PAGE, in this article, GPx bands have been referred to normal liver function is recommended over other HAas GPx1–GPx 4 based on their relative migration in non- models with acute liver failure [37]. This is because thedenaturing 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 associatedlanes, 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 relevantin vitro in the cell extracts from the respective brain re- for interpreting the data obtained using HA animal modelsgions (unpublished results). Contrary to this, in comparison [24]. In the present report, we have used hyperammonemicto the samples from control rats, though there was a small rats induced by administration of ammonium acetatedecline (P < 0.05) in the activity of GR in the cerebellum, wherein, as reported earlier [24], ~5–7 and 1.5–1.8 foldGR activity in the cerebral cortex remained unchanged increases in brain ammonia level was considered as acuteduring acute HA. and chronic HA groups respectively. Brain processes ~20% of O2 consumed by the wholeEffect of chronic HA on antioxidant enzymes body for generating ATP via oxidative phosphorylation in mitochondria and therefore, brain cells are consistentlyFigures 5–7 illustrate that in cerebral cortex of chronic HA exposed to high ROS. Abundance of myelinated nerverats, as compared to the control group, activities as well as fibers makes brain enriched with phospholipids containinglevels of active fractions of SOD, catalase and all isoforms poly unsaturated fatty acids, and thus, brain cells becomeof 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 ofdeclined significantly (P < 0.05), there were no significant oxidative damage produced by ROS in the brain. The overchange observed in the activity and the level of catalase activation of NMDA receptors [1] and ammonia inducedduring chronic HA. Moreover, as compared to the control mitochondrial dysfunction [4, 5] could be the main sourcegroup 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 haveno 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 cerebellumDiscussion in pure HA rats, significantly increased level of lipid per- oxidation (~ 2 times higher) in cerebellum than the cortexIn the present article, we intended to address two aspects of under both acute and chronic conditions clearly suggestammonia neurotoxicity, one the relationship between the that cerebellum is more susceptible for oxidative damagedegree of HA and oxidative stress in brain & secondly, due to ammonia toxicity than the cortex. Furthermore, ~ 3since cerebellum associated functions are affected the most and 2 fold increases in the MDA level in cerebellum ofduring chronic HA, is it that cerebellum is more susceptible acute and chronic HA rats respectively suggest for a causeto ammonia toxicity than other brain regions with respect and effect relationship between the degree of HA in brainto oxidative stress. For such comparative studies on and the oxidative damage in cerebellum. Nonetheless, 123
  6. 6. 108 Neurochem Res (2008) 33:103–113A 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 extractsFig. 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 specificThe values in A represent mean ± SD where n = 4 and each development of catalase bands. The gel photograph in B is aexperiment done in duplicate. In case of B, pooled tissue extracts representative out of the 3 PAGE repeats. In panel C, relativefrom 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 3development of SOD bands. The gel photograph in B is a PAGE repeat experiments. ***P < 0.001 (control versus experimentalrepresentative out of the 4 PAGE repeats. In panel C, relative groups)densitometric values of SOD bands from experimental group as % ofthe control lane have been presented as mean ± SD from the 4 PAGErepeat experiments *P < 0.05, **P < 0.01, ***P < 0.001 (control brain regions under acute as well as chronic HA conditionsversus experimental groups) (Table 1). In the cellular antioxidant pathway, the turnover of GSH/GSSG is regulated by synchronized activities ofcortex showed resistance to HA dependent oxidative stress, GPx and GR in mammalian cells. Both these enzymes didas there was no change in MDA level in the cortex of not show much alternation, except a moderate decrease inchronic but with a mild (1.3 fold) increase in that from GPx and GR in cortex and cerebellum respectivelyacute 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 regionsresponsible to maintain reducing equivalents under oxida- during acute HA. However, significantly opposite trends oftive stress, is another critical factor to assess the level of GPx and GR in the cerebral cortex of chronic HA ratsoxidative stress in mammalian cells. Interestingly, there (Figs. 7, 8) did not correlate with the unchanged level ofwas no significant change in the level of GSH in both the GSH in the cortex of rats with chronic HA. It is suggested123
  7. 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 25GPx 3 * 20GPx 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 playprotein in cerebral cortex and cerebellum of rats. In case of A, pooled major role of antioxidant activities [19, 20]. With thetissue extracts from 4 rats containing 30 lg protein in each lane was increased production of ROS, most of these enzymes wereelectrophoressed on 10% non-denaturing PAGE followed by substrate found to be declined in whole brain of rat with acute HEspecific development of GPx bands. The gel photograph in A is arepresentative out of the 4 PAGE repeats. In panel B, relative [8]. However, according to the results presented here, whendensitometric values of GPx bands from experimental group as % of the levels of all these enzymes were compared in concertedthe control lane have been presented as mean ± SD from the 4 PAGE manner in two different brain regions (cerebral cortex andrepeat 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 degreethat 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 wastripeptide under unphysiological conditions in brain [41, a significant decline in the levels of SOD, catalase and GR42]. 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 lipidsusceptible 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 andin the cells, and this enzyme has been reported to be in- catalase, however, with a moderate decrease in GPx and nocreased in the astrocytes under acute HA condition [43]. change in the level of GR under acute HA (Figs. 3, 4). InThus, it is likely that these additional routes could con- view of relatively less increase in the level of lipidtribute for maintaining GSH level in the cortex of chronic peroxidation due to acute HA in the cortex (~ 2 times lessHA 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 preventtered level of GSH in cerebellum of chronic HA rats where, oxidative damage in cerebral cortex even at the face ofGPx 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 ofthe extent of oxidative damage produced by ROS during lipid peroxidation in cerebellum (Table 1), however, withneuropathology [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 GPxbalance the deleterious effects of ROS [44, 45]. Excess are mainly accountable to allow oxidative damage inof superoxide anion (O–), the major ROS produced in 2 cerebellum and unaffected GR plays a permissive role in 123
  8. 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 CatalaseSOD 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 eachFig. 5 Effect of chronic hyperammonemia on activity (A) and level experiment done in duplicates. In case of B, pooled tissue extractsof 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 specificexperiment done in duplicate. In case of B, pooled tissue extracts development of catalase bands. The gel photograph in B is afrom 4 rats containing 60 lg protein in each lane was electropho- representative out of the 3 PAGE repeats. In panel C, relativeressed 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 3representative out of the 4 PAGE repeats. In panel C, relative PAGE repeat experiments. *P < 0.05, ***P < 0.001 (control versusdensitometric values of SOD bands from experimental group as % of experimental groups)the control lane have been presented as mean ± SD from the 4 PAGErepeat 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 crucialmaintaining the normal level of GSH (Table 1) during metabolites which are normally produced otherwise duringchronic HA in this brain region. This again supports the acute HA [8]. At the face of significant decline in theview that SOD and GPx are the most critical antioxidant activity of most of the antioxidant enzymes, SOD activityenzymes in brain [19, 20]. Nonetheless, since, both these was reported to be increased significantly in all the brainenzymes declined specifically in cerebellum (as compared regions of rats with fulminate liver type acute HE [10]. Asto the cortex) and that cerebellum associated functions are per the results presented here, however, it is evident thataffected the most in HE patients [14, 15], it may be argued chronic HA produces adaptive changes only in cerebralthat 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 chronicaccountable 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 enzymecreases in the levels of SOD, catalase and GPx (Figs. 5–7) has a functionally distinct role, or cooperates with otherand 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. upplastic tissue so far metabolic adaptations are concerned regulation of SOD-GPx in cortex and their down regulation123
  9. 9. Neurochem Res (2008) 33:103–113 111 A 35 Control HA Control HA Control 30 HA GPx 1 GR (U/mg protein) 25 ** 20GPx 2 15GPx 3GPx 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+ inFig. 7 Effect of chronic hyperammonemia on level of active GPx brain is associated with Alzheimers type II astrocytosisprotein in cerebral cortex and cerebellum of rats. In case of A, pooledtissue extracts from 4 rats containing 30 lg protein in each lane was [50], a hall mark of acute HA [1] and as reviewed byelectrophoressed on 10% non-denaturing PAGE followed by substrate Takeda [51], increased level of Mn2+ inhibits catalase andspecific development of GPx bands. The gel photograph in A is a also induces a burst of H2O2 in brain cells. Also, as per therepresentative out of the 4 PAGE repeats. In panel B, relative results presented here (Fig. 2A), a drastic decrease in thedensitometric values of GPx bands from experimental group as % ofthe control lane have been presented as mean ± SD from the 4 PAGE activity of catalase in cerebellum of acute HA rats may alsorepeat 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 inin 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, itHA. Opposite responses of cortex and cerebellum to NO has been demonstrated that inhibition of SOD at cellulardependent signaling pathway during HA in rats [12] and level induces increase in the mRNA level of this enzymealso in HE patients [16] provide support to this argument. [52], and SOD proteins are highly resistant to denaturationSuch a pattern has been shown in other neurological & oxidative damage even at a high concentration of H2O2disorders also. Different antioxidant enzymes showed [48]. Therefore, it is likely that inactivation of SODdifferential alterations in the brain of patients with observed in cell free extracts due to increased oxidativeAlzheimers type dementia [21] and also in D-amphitamine burst in cerebellum of acute HA rats might not be reflectedinduced neurotoxicity [22]. Increase in the level of SOD at protein level (Fig. 1). Accordingly, since inactivationand catalase in different brain regions of rats with mala- of SOD would be expected to be minimal during mildthion-induced oxidative stress is another example of oxidative stress, in vitro activity data and level of SODadaptive 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 cerebralsignificant changes in the activities of SOD, catalase and cortex of acute HA rats (Fig. 1) with ~ 2 times lessGPx during HA, these enzymes were further analyzed on oxidative stress than cerebellum (Table 1, MDA data). AsPAGE. It was interesting to note that while level of SOD Mn2+ also inhibits catalase in the brain [51] and suchprotein 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 bein vitro) showed significant decline (Fig. 1A). Similar given for significant decreases in the activity of catalasepattern was observed with catalase in both the brain regions in vitro but with insignificant change in its level on PAGEof acute HA rats (Fig. 2A–C). Such a mismatch could be analysis in both the brain regions of acute HA rats. These 123
  10. 10. 112 Neurochem Res (2008) 33:103–113arguments get further support from a uniform correlative 11. Hermenegildo C, Montoliu C, Llansola M et al (1998) Chronicpattern 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 10:3201–3209HA rats (Figs. 5, 6) showing significantly less oxidative 12. Rodrigo R, Felipo V (2006) Brain regional alternations in thestress as compared to the acute HA rats (Table 1, MDA modulation of the glutamate-nitric oxide-cGMP pathway in liverdata). Thus, it is evident that the extent of oxidative stress cirrhosis: role of hyperammonemia and cell types involved. Neorochem Int 48:472–477induced during acute HA acts as an additional factor in 13. 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