http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2014.945108
ORIGINAL ARTICLE
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Hesperidin restores experimentally induced neurotoxicity in Wistar rats Mehar Naseem and Suhel Parvez Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi, India
Abstract
Keywords
Carbon tetrachloride (CCl4) is a highly toxic industrial solvent with pronounced systemic toxicity including brain. Neurotoxicity may be a direct result of hepatic dysfunction from CCl4 intoxication. Over the years CCl4 has been used as an excellent model for studying experimentally induced neurotoxicity in murine models. Hesperidin (HP) is a known cytoprotectant with comprehensive anti-oxidant and neuroprotective properties. The aim of the present study was to evaluate experimentally induced neurotoxicity by CCl4 and its abrogation by using antioxidant potential of HP. CCl4 caused a significant enhancement in the lipid peroxidation (LPO) levels and protein carbonyl (PC) content. HP supplementation significantly restored the LPO levels and PC content. It also replenished the altered enzymatic and non-enzymatic antioxidants in brain tissues of rats. The neurotoxicity markers were also restored to normalcy with HP treatment. It is suggested that HP, by attenuating neuronal oxidative stress, holds promise that can ameliorate CCl4-induced neurotoxicity. HP has the potential to be explored as a universal neuroprotectant in xenobiotically induced neurotoxicity mediated by oxidative stress.
Biomarkers, brain, carbon tetrachloride, natural compound, oxidative stress, rodents
Introduction A large increase in reactive oxygen species (ROS) which are produced during various cellular metabolism leads to several damaging effects like tissue damage, DNA damage, oxidative stress, lipid peroxidation (LPO), and cell death (Saquib et al., 2012). ROS is implicated in pathogenesis of a number of neurodegenerative diseases in brain, cognitive dysfunction, cancer, and a variety of other conditions (Kovacic & Somanathan, 2012). Brain also contains high level of ascorbate and iron, which may be essential during brain development involved in the production of ROS. Numerous studies have indicated that oxidative stress is responsible for the disruption of blood brain barrier (BBB) integrity associated with neurodegenerative disorders as it is prone to ROS (Freeman & Keller, 2012). Brain may deal in a different way with oxidative stress as compared with liver or kidney (Seminotti et al., 2012). Carbon tetrachloride (CCl4) is a well-known hepatotoxic agent which has also been implicated in inhibiting mitochondrial respiratory chain in the brain (Boer et al., 2009). Natural compounds have been used for various purposes in medicines,
Address for correspondence: Dr. Suhel Parvez, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110062, India. Tel: +91 11 26059688x5573. Fax: +91 11 26059663. E-mail: [email protected], sparvez@ jamiahamdard.ac.in
History Received 14 April 2014 Revised 15 June 2014 Accepted 11 July 2014 Published online 30 July 2014
cosmetics, and also as dietary supplements (Albarracin et al., 2012). Flavonoids are compounds, which are antioxidants from natural origin and are present in a number of natural sources (Kamaraj et al., 2009). Hesperidin (HP, 3,5,7-trihydroxy flavanone-7-rhamnoglucoside) is a biologically and pharmacologically active citrus bioflavonoid by-product of citrus cultivation, abundantly found in sweet orange and lemon (Gaur et al., 2011). It has been previously reported that HP is having free radical scavenging as well as anti-LPO properties and has been shown to be powerful against ROS and it has also been assayed for its neuroprotective efficacy in diabetic rat model (Ibrahim, 2008). It has attracted particular interest due to its several health beneficial effects like anti-inflammatory, anticarcinogenic, antimicrobial, and immunomodulatory effects (Nones et al., 2010). HP has been shown to be protective against various neurobehavioral alterations caused (Viswanatha et al., 2012). It has been well documented that HP is a potential neuroprotective antioxidant, it may because of being lipophilic in nature, crosses the BBB easily (Salem et al., 2012). HP deals with neurotoxicity due to its antioxidative and antiinflammatory properties and also protects the dopamine depletion which enhances it neuroprotective efficiency (Tamilselvam et al., 2013). However, protective role of HP against CCl4 induced neurotoxicity has not been investigated in a rodent model. The aim of the present study was to explore modulatory effects of HP against CCl4-induced neurotoxicity in Wistar rats.
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Toxicol Mech Methods, Early Online: 1–8
Materials and methods
Sample preparation
Chemicals
Post-mitochondrial supernatant (PMS) and homogenate preparation
Sulfosalicylic acid and thiobarbituric acid (TBA) were obtained from Hi-Media Labs (Mumbai, India). Butylated hydroxytoluene (BHT), o-phosphoric acid (OPA), trichloroacetic acid (TCA), 1-Amino-2-naphthol-4-sulfonic acid (ANSA), 2,4-dinitrophenylhydrazine (DNPH), and perchloric acid (PCA) were purchased from Merck Limited (Mumbai, India). Bovine serum albumin, reduced glutathione (GSH), oxidized glutathione (GSSG), and benzylamine hydrochloride (BAHC) were purchased from Sigma Chemicals Co. (St. Louis, MO). Reduced nicotinamide adenine dinucleotide phosphate (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), ethylenediaminetetraacetic acid (EDTA), and acetylthiocholine iodide (ATC) were obtained from SRL (Mumbai, India). Other routine chemicals were obtained from Hi-Media Labs (Mumbai, India), Merck Limited (Mumbai, India), and SRL (Mumbai, India). CCl4 and HP were obtained from Merck Limited (Mumbai, India) and SRL Labs (Mumbai, India), respectively. Animals Adult male Wistar rats weighing 250–300 g were used for the study. The experimental rats were acclimatized for a week prior to the commencement of the dose regime. The animals were housed under standard laboratory conditions where temperature was maintained at 22 ±2 C with a relative humidity of 65 ± 10% and a photoperiod of 12 h light/dark cycle. All the experiments were carried out according to the standard guidelines of Institutional Animal Ethics Committee (IAEC) of Jamia Hamdard (Hamdard University).
The brain of the animals was promptly excised and washed with chilled phosphate-buffered saline for the removal of blood. The brain was homogenized 1:10 w/v in 0.1 M sodium phosphate buffer (pH 7.4) with a Potter–Elvehjem homogenizer. The homogenates obtained were used to estimate brain TBARS. The homogenates were centrifuged at 10 500 g for 20 min at 4 C to get the PMS, using a REMI C-24 centrifuge (Remi Sales and Engineering, Ltd., Mumbai, India). The PMS supernatant obtained was used for the analysis of oxidative stress biomarkers, non-enzymatic antioxidants, enzymatic antioxidants, and neurotoxicity biomarkers. Biochemical estimation Oxidative stress biomarkers Determination of LPO. The extent of LPO was measured
using the procedure of Tabassum et al. (2007). The concentration of thiobarbituric acid reactive substances (TBARS) was determined using an extinction coefficient of 1.56 105 M1 cm1 and results were expressed as nmol TBARS formed/h/mg protein. Protein oxidation in brain PMS was measured as a concentration of protein carbonyls (PC) formed. PC level was determined using DNPH assay according to the procedure of Chaudhary & Parvez (2012). The results were expressed as nmol DNPH incorporated/mg protein based on the molar extinction coefficient of 2.1 104 M1 cm1.
Determination of protein oxidation.
Non-enzymatic antioxidants Experimental design
Estimation of non-protein thiol (NP-SH). The NP-SH was
The animals were randomly divided into four groups, and each group consisted of seven animals. The description about the different groups is as follows: Group 1: Control group. The animals were treated with a vehicle for HP (i.e. normal saline) by oral route for eight consecutive days and a single subcutaneous (s.c.) injection of olive oil on the 7th day. Group 2: HP group. In this group, rats received HP (200 mg/ kg b.w., oral) treatment for a period of 8 d. Group 3: CCl4 group. The animals were administered normal saline for 8 d and were exposed to CCl4 (2 ml/kg b.w., s.c., 40% v/v in olive oil) on the 7th day of the treatment. Group 4: HP (200 mg/kg b.w., oral) + CCl4 (2 ml/kg b.w., s.c.). Rats were given HP treatment for 8 d and a single injection of CCl4 on the 7th day along with HP. The dose of CCl4 and HP used in our study was based on previously published research reports (El-Sayed et al., 2008; Jayakumar et al., 2008). At the end of the experimental period of 8 d, the animals were anesthetized and sacrificed by decapitation. The brain was immediately dissected, and washed in chilled phosphate buffer to remove blood. The effect of CCl4 and role of HP on the oxidative stress biomarkers were studied in the brain tissue.
measured according to the method described by Govil et al. (2012). The molar extinction coefficient of 13 100M1 cm1 at 412 nm was used for the determination of the NP-SH content. The values were expressed as nmol of the NP-SH/mg protein. Estimation of reduced glutathione (GSH). The GSH content
was estimated according to the method of Chaudhary & Parvez (2012). The GSH concentration was calculated as nmol GSH/mg protein using a molar extinction coefficient of 1.36 104 M1 cm1. Enzymatic antioxidants The activity of GPx was assayed according to the method of Haque et al. (2003). The assay mixture consisted of sodium phosphate buffer (0.1 M, pH 7.4), EDTA (1 mM), sodium azide (1 mM), GSH (1 mM), NADPH (0.02 mM), H2O2 (0.25 mM), and 0.1 ml of PMS of brain in a total volume of 2 ml. Oxidation of NADPH was recorded kinetically at 340 nm. The enzyme activity was calculated as nmol NADPH oxidized/min/mg protein, using a molar extinction coefficient of 6.22 103 M1 cm1.
Activity of glutathione peroxidase (GPx).
The activity of GR was assayed by the method of Haque et al. (2003). The assay
Activity of glutathione reductase (GR).
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DOI: 10.3109/15376516.2014.945108
system consisted 0.1 M phosphate buffer (pH 7.4), 0.5 mM EDTA, 1 mM GSSG, 0.1 mM NADPH, and 0.3 ml supernatant of brain tissue in a total volume of 2 ml. The enzyme activity was quantitated at 25 C by measuring the disappearance of NADPH at 340 nm, and was calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.223 103 M1 cm1.
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homogenate when compared with the control group (Figure 1A). HP (200 mg/kg, oral) pre-treatment revealed a significant (p50.01) depletion in the LPO content of brain homogenate as compared with the CCl4-treated group. HP alone did not show any significant change in the LPO content of animals beyond control values. Effect on protein oxidation (PC)
The activity of GST was assayed using the method of Haque et al. (2003) by the catalytic reaction of CDNB with the sulfhydryl group of glutathione and was measured kinetically at 340 nm. The assay system consisted of 0.1 M phosphate buffer (pH 7.4), 10 mM GSH, 10 mM CDNB, and 0.1 ml of PMS of rat brain. The enzymatic activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar coefficient of 9.6 103 M1 cm1.
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Activity of glutathione-s-transferase (GST).
Exposure to CCl4 resulted in a significant (p50.001) enhancement in the activity of protein oxidation in the brain PMS of CCl4-treated animals as compared with the control group (Figure 1B). In HP pre-treated group, there was a significant (p50.01) depletion in the PC content of brain PMS. However, no significant effect in the PC content of HP treated group was observed when compared with control. Non-enzymatic antioxidant status
Neurotoxicity biomarkers
Effect on NP-SH
Activity of monoamine oxidase (MAO). The activity of MAO
The level of NP-SH in brain PMS significantly (p50.001) increased in CCl4-administered group of animals when compared with the animals of the control group (Figure 2A). CCl4-exposed HP pre-administered animals showed a significant (p50.001) decrease in the NP-SH level as compared with that of CCl4-administered animals.
was measured by using the method of Chaudhary & Parvez (2012). The enzyme activity was calculated as mmol BAHC hydrolyzed/min/mg protein using a molar extinction coefficient of 7.6925 M1 cm1. Activity of acetylcholinesterase (AChE). The activity of AChE
was estimated by using the method of Chaudhary & Parvez (2012). The enzyme activity was calculated as nmol ATC hydrolyzed/min/mg protein using a molar extinction coefficient of 1.36 104 M1 cm1. The activity of total ATPase was measured as the release of inorganic phosphate (Pi) by the method of Chaudhary & Parvez (2012). The activity was measured as mg Pi liberated/min/mg protein.
Activity of total ATPase.
Determination of protein Estimation of protein contents in biological fractions of brain was done using bovine serum albumin as standard by the method of Lowry et al. (1951). Statistical analysis The results obtained were expressed as mean ± standard error (SE). All data were analyzed using analysis of variance (ANOVA) followed by Tukey’s test. Values of p50.05 were considered as significant. All the statistical analyses were performed using graph pad prism 5 software (Graph Pad Software, Inc., San Diego, CA).
Results No mortality and significant alteration in the body weight of different groups of subject/animals were documented during and after the treatment (data not shown). Oxidative stress biomarkers Effect on LPO Treatment of animals with CCl4 (2 ml/kg b.w.) showed a significant (p50.001) elevation in the LPO content of brain
Figure 1. Effect of CCl4 and HP on (A) LPO and (B) PC in brain of rat. Values were expressed as nmoles of TBARS formed/h/mg protein and nmoles of DNPH incorporated/mg protein, respectively. Each value represented as mean ± SE (n ¼ 6). Significant differences were indicated by ***p50.001 when compared with control and significant difference were indicated by ##p50.01 when compared with CCl4-treated animals.
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CCl4 significantly increased (p50.001) the glutathione reductase level (GR) in CCl4-treated group as compared with the control group (Figure 3B). A single dose of CCl4 combined with pretreatment of HP showed a significant (p50.01) decrease in GR activity as compared with the CCl4intoxicated group. HP treatment showed no significant change in the activity of GR in the PMS of brain. CCl4 exposure showed a significant (p50.001) rise in the activity of GST when compared with the control group (Figure 3C). In a single dose of CCl4 in the HP pre-treated group revealed a significant (p50.001) decline in the GST activity in brain PMS as compared with the CCl4-treated group. Group treated with HP showed no effect in the activity of GST. Effect on neurotoxicity biomarkers
Figure 2. Effect of CCl4 and HP on level of (A) NP-SH and (B) GSH in brain tissue of rat. Values were expressed as nmoles NP-SH/mg protein and nmoles GSH/mg protein, respectively. Each value represented as mean ± SE (n ¼ 6). Significant differences were indicated by ***p50.001 when compared with control. Significant differences ###p50.001 were shown when compared with CCl4-treated animals.
HP-treated animals showed no any significant alteration in the NP-SH level in brain PMS when compared with control group of animals. Effect on GSH Significant (p50.001) higher level of the GSH content was observed in brain PMS of CCl4-exposed animals when compared with the control group (Figure 2B). HP pretreatment prior to CCl4 injection significantly (p50.001) declines the GSH content and prevented these elevations. Animals treated with HP did not show any significant change when compared with the control group. Enzymatic antioxidants activities Effect on glutathione-metabolizing enzymes There was a significant (p50.001) elevation in the activity of GPx in brain PMS in the CCl4-treated group as compared with the control group (Figure 3A). HP pretreatment showed a significant (p50.001) decline in the activity of GPx as compared with the CCl4-treated group. HP-treated group did not change the GPx level in the brain PMS when compared with the control group.
There was a significant (p50.001) increase in the activity of MAO in the CCl4-treated group of animals (Figure 4A). HP supplementation along with a single dose of CCl4 showed a significant (p50.001) decline in the activity of MAO as compared with that of the CCl4-treated group. No effect in the activity of MAO was seen in the HP-treated group. The CCl4-treated group showed significant (p50.001) rise in the activity of AChE as compared with the control group (Figure 4B). The HP pre-exposed group significantly (p50.001) decreased the activity of AChE when compared with the CCl4-treated group and prevented such elevations. The HP-treated group exhibited no remarkable difference when compared with control. A single dose of CCl4 caused a significant (p50.01) increase in the total ATPase content in brain PMS of animals when compared with control (Figure 4C). HP pre-treatment in CCl4-treated group caused significant (p50.01) decline in the activity of ATPase. The HP-treated group did not show significant change in the activity.
Discussion Rats intoxicated with CCl4 leads to the generation of ROS, ultimately resulting in neurotoxicity in rats (Samudram et al., 2009). These highly reactive free radicals of CCl4 generated by the CYP are capable of covalently binding to macromolecules and cause intense LPO leading to the degeneration of cell membrane of endoplasmic reticulum which is eminently rich in PUFA (Altas et al., 2011). HP possesses highest reducing power and exhibits anti-oxidative properties (Salem et al., 2012). TBARS is a by-product of LPO, considered as a reliable index of tissue damage (Tabassum et al., 2007). In the present study, increased levels of TBARS in brain tissue may be due to increased production of these free radicals which are unstable and highly active, leading to fatty acid damage following CCl4 intoxication which will lead to enhanced LPO. The potential amelioration of free radicals which leads to fatty acid damage by HP, which is a wellknown antioxidant reducing the severity of LPO by decreasing TBARS concentration. It suggested that HP exerts its protective effect by regulating LPO of membranes by scavenging the debilitating consequences of free radicals and augmenting the antioxidant defence system (Pradeep et al., 2008).
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DOI: 10.3109/15376516.2014.945108
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Figure 3. Effect of CCl4 and HP on (A) GPx, (B) GR and (C) GST activity in brain of rat. GPx and GR activities were expressed as nmoles of NADPH oxidized/min/mg protein whereas GST activity was expressed as nmoles of CDNB conjugate formed/min/mg protein. Each value represented as mean ± SE (n ¼ 6). Significant differences were shown by ***p50.001 when compared with control. Significant differences were indicated by ##p50.01 and ###p50.001 when compared with CCl4-treated animals.
PC is considered as an excellent biomarker of oxidative protein damage as proteins are susceptible to oxidative modification (Dalle-Donne et al., 2003). Oxidative modification of proteins changes its configuration via aggregation, fragmentation, and formation of cross linkages in the polypeptide chain and also leads to secondary impairment of biomolecules with loss of protein biological function, leads to the generation of superoxide radical (Chen et al., 2005). Addition of carbonyl group in proteins is directly proportional to the protein subjected to oxidative damage and an increase in carbonyl content is associated with various pathological disorders (Almroth et al., 2005). A significant increase in protein oxidation was seen in the brain of animals with the single-dose administration of CCl4 as compared with the control group. It might be due to increased oxidatively modified protein in brain with the administration of CCl4. Our result is in agreement with the findings of previous result performed with liver (Srivastava & Shivanandappa, 2010). Interestingly, pre-administration of HP attenuated the effect of CCl4-induced protein damage, indicating that HP has a protective action against CCl4-induced neurotoxicity. Our results are in corroboration with the recent finding where HP has been shown to be a good natural antioxidant against cisplatin-induced oxidative damage (Sahu et al., 2013). HP has been found to reduce the PC content in our study thereby reducing oxidative stress and oxidative damage of protein by
scavenging the free radicals causing modification in the protein structure. Dietary antioxidant supplementation such as bioflavonoids mainly protects cells and organs against ROS-induced oxidative damage (Tsai et al., 2013). GSH scavenges free radicals and provides a defence system against ROS as well as detoxifies various xenobiotic compounds, H2O2, lipid hydroperoxides, and electrophilic compounds (Manna et al., 2011). It plays crucial role in defence mechanism and also acts as a co-enzyme for numerous enzymes involved in the cellular defence system (Masella et al., 2005). GPx constitutes a family of enzyme which reduces H2O2 and lipid hydroperoxides to the corresponding stable hydroxy compounds and H2O utilizing GSH as a reducing substrate (Dickinson & Forman, 2002). GR is a flavoenzyme and is represented by a single copy gene in humans which takes part in the reduction of GSSG (oxidized glutathione) to GSH with the help of NADPH (Li et al., 2010). The activity of GR is very important to maintain the level of GSH/GSSG ratio. GST is a well-known phase-2 enzyme which plays a very crucial role in the detoxification process of electrophilic compounds, favoring the elimination from the body of the organ by conjugation with glutathione (Afzal et al., 2013). GSH oxidizes to GSSG by peroxide and GPx. GSH is restored from GSSG by the enzyme GR using NADPH as an electron donor and the ratio of GSH/GSSG within a cell is considered as the measure of
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Figure 4. Effect of CCl4 and HP on (A) MAO, (B) AChE, and (C) total ATPase activity in brain of rat. MAO activity was expressed as mmol BAHC hydrolyzed/min/mg protein, AChE activity as nmol ATC hydrolyzed/min/mg protein and total ATPase activity as mg Pi liberated/min/mg protein. Each value represented as mean ± SE (n ¼ 6). Significant differences were shown by **p50.01 and ***p50.001 when compared with control. Significant differences were indicated by ##p50.01 and ###p50.001 when compared with CCl4-treated animals.
cellular toxicity (Board & Menon, 2013). An increase in the activity of GR, GPx, and GST was found in the CCl4intoxicated group of animals. An increase in the activity of glutathione-dependent enzymes reveals the primary response of the cell to the exposure to oxidative stress-inducing toxic compounds with the potential of inducing oxidative stress and damage caused in the cells and the cells may reduce the severity of oxidative damage by maintaining a reduced state in the cell. Induction in the activity of GPx on CCl4 administration suggests that there might be a rapid oxidation of GSH mediated by peroxide. This result is in agreement with the findings of the effect of cadmium (Cd) on different organs, liver, kidney, and gills of freshwater murrel Channa punctatus (Dabas et al., 2012). The increased level of GST in CCl4intoxicated animals suggests that it might be due to the conjugation between trichloromethyl radical and GSH to mitigate the effect of increased oxidative stress. Our result is in agreement with the result obtained earlier in the brain of the CCl4-treated group of animals (Soliman & Fahmy, 2011). HP administration restored the increased level of GSH which suggests the reversal of the increased oxidative stress. Our result corroborates with the findings of antioxidative and antiinflammatory role of HP on the rat air pouch model of inflammation (Jain & Parmar, 2011). It may be due to the depletion in the level of cysteine with the pretreatment of HP as cysteine which is one of the major constituents of tripeptide
antioxidant GSH which may affect the level of GSH in the HP pre-treated CCl4-intoxicated group of animals. MAO, AchE, and Na+/K+-ATPase are considered as important neurotoxicity biomarkers. MAO is a flavincontaining brain-specific enzyme which plays an important role in oxidative deamination of endogenous monoamine neurotransmitters, regulates the oxidative deamination of endogenous and exogenous monoamines in the central nervous system, and converts biogenic amines to their corresponding aldehydes (Secci et al., 2011). Alteration in the function of MAO may lead to age-related disorder and a number of neurological disorders and depression (Meyer et al., 2006). MAO takes part in the metabolism of catecholamine like adrenaline, noradrenaline, and dopamine into free radicals, abundance of which may be responsible for serotonin syndrome (Ghareeb et al., 2011). An increase in the activity of MAO suggests that there is a high level of oxidative stress. In the present assessment, a single dose of CCl4 was found to be effective in increasing the activity of MAO. Pretreatment of animals with HP inhibited the activity of MAO. HP was found to be an effective inhibitor of MAO as it inhibited the metabolism of the monoamines metabolization of which form hydrogen peroxide and ammonia which is the potential toxic products of MAO. AchE is a key enzyme in the nervous system which is responsible for the degradation of neurotransmitter,
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acetylcholine (Ach), during neurotransmission which is necessary for learning and memory deficits (Parent & Baxter, 2004) and also plays a very important role in the cholinergic activity (Antoniades et al., 2002). Our result is in accordance with a recent finding in which an increase in the activity of AchE in the region of brain has been reported with streptozotocin treatment (Biasibetti et al., 2013). An increase in the activity of AchE in the brain is responsible for the low availability of acetylcholine and, therefore, it may lead to the alteration in cholinergic transmission (Tiwari et al., 2009). HP pre-treatment was able to reverse the altered activity of AchE which deals with the cognitive defect by increasing cholinergic neurotransmission as reported in a report with a natural antioxidant curcumin (Agrawal et al., 2010). The sodium potassium adenosine triphosphate (Na+/K+ATPase) is an enzyme which maintains neuronal excitability and plays a major role in the export and import of three Na+ ions out and two K+ ions in of the cell by utilizing ATP across the plasma membrane against their potential gradient in the nervous system and Na+/K+-ATPase has also been reported as a signal transducer (Lingrel, 2010). The Na+/K+-ATPase is an integral membrane heterodimer belongs to a superfamily of proteins collectively designated as P-type ATPase family (Jaitovich & Bertorello, 2006). In the present study, a remarkable elevation is shown in the activity of Na+/K+ATPase in the CCl4-intoxicated group of rats. HP pretreatment suppressed the elevated level of Na+/K+-ATPase activity in brain due to its neuroprotective nature. Our result is in agreement with the previous result reported with some natural compounds, green tea and bamboo leaves on Na efflux channel in ovariectomized rats (Ryou et al., 2012). Further studies are required for deciphering cellular and molecular mechanisms of experimentally induced neurotoxicity and the role of HP in its attenuation.
Declaration of interest The authors declare that they have no conflict of interest. University Grants Commission (UGC), Government of India, is gratefully acknowledged for providing funding under Faculty Research Award to S.P. M.N. was supported by a Junior Research fellowship from UGC – Maulana Azad National Fellowship program.
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acetylcholinesterase activity in a streptozotocin-induced model of dementia. Behav Brain Res 236:186–93. Board PG, Menon D. (2013). Glutathione transferases, regulators of cellular metabolism and physiology. Biochim Biophys Acta 1830: 3267–88. Boer LA, Panatto JP, Fagundes DA, et al. (2009). Inhibition of mitochondrial respiratory chain in the brain of rats after hepatic failure induced by carbon tetrachloride is reversed by antioxidants. Brain Res Bull 80:75–8. Chaudhary S, Parvez S. (2012). An in vitro approach to assess the neurotoxicity of valproic acid-induced oxidative stress in cerebellum and cerebral cortex of young rats. Neuroscience 225:258–68. Chen YR, Chen CL, Zhang L, et al. (2005). Superoxide generation from mitochondrial NADH dehydrogenase induces self-inactivation with specific protein radical formation. J Biol Chem 280:37339–48. Dabas A, Nagpure NS, Kumar R, et al. (2012). Assessment of tissuespecific effect of cadmium on antioxidant defense system and lipid peroxidation in freshwater murrel, Channa punctatus. Fish Physiol Biochem 38:469–82. Dalle-Donne I, Giustarini D, Colombo R, et al. (2003). Protein carbonylation in human diseases. Trends Mol Med 9:169–76. Dickinson DA, Forman HJ. (2002). Cellular glutathione and thiols metabolism. Biochem Pharmacol 64:1019–26. El-Sayed el SM, Abo-Salem OM, Abd-Ellah MF, Abd-Alla GM. (2008). Hesperidin, an antioxidant flavonoid, prevents acrylonitrileinduced oxidative stress in rat brain. J Biochem Mol Toxicol 22: 268–73. Freeman LR, Keller JN. (2012). Oxidative stress and cerebral endothelial cells: regulation of the blood-brain-barrier and antioxidant based interventions. Biochim Biophys Acta 1822:822–9. Gaur V, Aggarwal A, Kumar A. (2011). Possible nitric oxide mechanism in the protective effect of hesperidin against ischemic reperfusion cerebral injury in rats. Indian J Exp Biol 49:609–18. Ghareeb DA, Hafez HS, Hussien HM, Kabapy NF. (2011). Nonalcoholic fatty liver induces insulin resistance and metabolic disorders with development of brain damage and dysfunction. Metab Brain Dis 26:253–67. Govil N, Chaudhary S, Waseem M, Parvez S. (2012). Postnuclear supernatant: an in vitro model for assessing cadmium-induced neurotoxicity. Biol Trace Elem Res 146:402–9. Haque R, Bin-Hafeez B, Parvez S, et al. (2003). Aqueous extract of walnut (Juglans regia L.) protects mice against cyclophosphamide induced biochemical toxicity. Hum Exp Toxicol 22:473–80. Ibrahim SS. (2008). Protective effect of hesperidin, a citrus bioflavonoid, on diabetes-induced brain damage in rats. J Appl Sci Res 4:84–95. Jain M, Parmar HS. (2011). Evaluation of antioxidative and antiinflammatory potential of hesperidin and naringin on the rat air pouch model of inflammation. Inflamm Res 60:483–91. Jaitovich AA, Bertorello AM. (2006). Na+, K+-Atpase: an indispensable ion pumping-signaling mechanism across mammalian cell membranes. Semin Nephrol 26:386–92. Jayakumar T, Sakthivel M, Thomas PA, Geraldine P. (2008). Pleurotus ostreatus, an oyster mushroom, decreases the oxidative stress induced by carbon tetrachloride in rat kidneys, heart and brain. Chem Biol Interact 176:108–20. Kamaraj S, Ramakrishnan G, Anandakumar P, et al. (2009). Antioxidant and anticancer efficacy of hesperidin in benzo(a)pyrene induced lung carcinogenesis in mice. Invest New Drugs 27:214–22. Kovacic P, Somanathan R. (2012). Redox processes in neurodegenerative disease involving reactive oxygen species. Curr Neuropharmacol 10: 289–302. Li ZH, Zlabek V, Grabic R, et al. (2010). Modulation of glutathionerelated antioxidant defense system of fish chronically treated by the fungicide propiconazole. Comp Biochem Physiol 152:392–8. Lingrel JB. (2010). The physiological significance of the cardiotonic steroid/ouabain-binding site of the Na+,K+-ATPase. Annu Rev Physiol 72:395–412. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–75. Manna P, Bhattacharyya S, Das J, et al. (2011). Phytomedicinal role of pithecellobium dulce against CCl4-mediated hepatic oxidative impairments and necrotic cell death. Evid Based Complement Alternat Med 2011:832805. Masella R, Di Benedetto R, Varı` R, et al. (2005). Novel mechanisms of natural antioxidant compounds in biological systems: involvement of
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Secci D, Bolasco A, Chimenti P, Carradori S. (2011). The state of the art of pyrazole derivatives as monoamine oxidase inhibitors and antidepressant/anticonvulsant agents. Curr Med Chem 18:5114–44. Seminotti B, da Rosa MS, Fernandes CG, et al. (2012). Induction of oxidative stress in brain of glutaryl-CoA dehydrogenase deficient mice by acute lysine administration. Mol Genet Metab 106:31–8. Soliman AM, Fahmy SR. (2011). Protective and curative effects of the 15 KD isolated protein from the Peganum harmala L. seeds against carbon tetrachloride induced oxidative stress in brain, tests and erythrocytes of rats. Eur Rev Med Pharmacol Sci 15:888–99. Srivastava A, Shivanandappa T. (2010). Hepatoprotective effect of the root extract of Decalepis hamiltonii against carbon tetrachlorideinduced oxidative stress in rats. Food Chem 118:411–17. Tabassum H, Parvez S, Rehman H, et al. (2007). Nephrotoxicity and its prevention by taurine in tamoxifen induced oxidative stress in mice. Hum Exp Toxicol 26:509–18. Tamilselvam K, Nataraj J, Janakiraman U, et al. (2013). Antioxidant and anti-inflammatory potential of hesperidin against 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine-induced experimental Parkinson’s disease in mice. Int J Nutr Pharmacol Neurol Dis 3:294–302. Tiwari V, Kuhad A, Bishnoi M, Chopra K. (2009). Chronic treatment with tocotrienol, an isoform of vitamin E, prevents intracerebroventricular streptozotocin-induced cognitive impairment and oxidative– nitrosative stress in rats. Pharmacol Biochem Behav 93:183–9. Tsai CF, Hsu YW, Ting HC, et al. (2013). The in vivo antioxidant and antifibrotic properties of green tea (Camellia sinensis, Theaceae). Food Chem 136:1337–44. Viswanatha GL, Shylaja H, Sandeep Rao KS, et al. (2012). Hesperidin ameliorates immobilization-stress-induced behavioral and biochemical alterations and mitochondrial dysfunction inmice bymodulating nitrergic pathway. ISRN Pharmacol 2012:479570.
ORIGINAL ARTICLE
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Hesperidin restores experimentally induced neurotoxicity in Wistar rats Mehar Naseem and Suhel Parvez Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi, India
Abstract
Keywords
Carbon tetrachloride (CCl4) is a highly toxic industrial solvent with pronounced systemic toxicity including brain. Neurotoxicity may be a direct result of hepatic dysfunction from CCl4 intoxication. Over the years CCl4 has been used as an excellent model for studying experimentally induced neurotoxicity in murine models. Hesperidin (HP) is a known cytoprotectant with comprehensive anti-oxidant and neuroprotective properties. The aim of the present study was to evaluate experimentally induced neurotoxicity by CCl4 and its abrogation by using antioxidant potential of HP. CCl4 caused a significant enhancement in the lipid peroxidation (LPO) levels and protein carbonyl (PC) content. HP supplementation significantly restored the LPO levels and PC content. It also replenished the altered enzymatic and non-enzymatic antioxidants in brain tissues of rats. The neurotoxicity markers were also restored to normalcy with HP treatment. It is suggested that HP, by attenuating neuronal oxidative stress, holds promise that can ameliorate CCl4-induced neurotoxicity. HP has the potential to be explored as a universal neuroprotectant in xenobiotically induced neurotoxicity mediated by oxidative stress.
Biomarkers, brain, carbon tetrachloride, natural compound, oxidative stress, rodents
Introduction A large increase in reactive oxygen species (ROS) which are produced during various cellular metabolism leads to several damaging effects like tissue damage, DNA damage, oxidative stress, lipid peroxidation (LPO), and cell death (Saquib et al., 2012). ROS is implicated in pathogenesis of a number of neurodegenerative diseases in brain, cognitive dysfunction, cancer, and a variety of other conditions (Kovacic & Somanathan, 2012). Brain also contains high level of ascorbate and iron, which may be essential during brain development involved in the production of ROS. Numerous studies have indicated that oxidative stress is responsible for the disruption of blood brain barrier (BBB) integrity associated with neurodegenerative disorders as it is prone to ROS (Freeman & Keller, 2012). Brain may deal in a different way with oxidative stress as compared with liver or kidney (Seminotti et al., 2012). Carbon tetrachloride (CCl4) is a well-known hepatotoxic agent which has also been implicated in inhibiting mitochondrial respiratory chain in the brain (Boer et al., 2009). Natural compounds have been used for various purposes in medicines,
Address for correspondence: Dr. Suhel Parvez, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110062, India. Tel: +91 11 26059688x5573. Fax: +91 11 26059663. E-mail: [email protected], sparvez@ jamiahamdard.ac.in
History Received 14 April 2014 Revised 15 June 2014 Accepted 11 July 2014 Published online 30 July 2014
cosmetics, and also as dietary supplements (Albarracin et al., 2012). Flavonoids are compounds, which are antioxidants from natural origin and are present in a number of natural sources (Kamaraj et al., 2009). Hesperidin (HP, 3,5,7-trihydroxy flavanone-7-rhamnoglucoside) is a biologically and pharmacologically active citrus bioflavonoid by-product of citrus cultivation, abundantly found in sweet orange and lemon (Gaur et al., 2011). It has been previously reported that HP is having free radical scavenging as well as anti-LPO properties and has been shown to be powerful against ROS and it has also been assayed for its neuroprotective efficacy in diabetic rat model (Ibrahim, 2008). It has attracted particular interest due to its several health beneficial effects like anti-inflammatory, anticarcinogenic, antimicrobial, and immunomodulatory effects (Nones et al., 2010). HP has been shown to be protective against various neurobehavioral alterations caused (Viswanatha et al., 2012). It has been well documented that HP is a potential neuroprotective antioxidant, it may because of being lipophilic in nature, crosses the BBB easily (Salem et al., 2012). HP deals with neurotoxicity due to its antioxidative and antiinflammatory properties and also protects the dopamine depletion which enhances it neuroprotective efficiency (Tamilselvam et al., 2013). However, protective role of HP against CCl4 induced neurotoxicity has not been investigated in a rodent model. The aim of the present study was to explore modulatory effects of HP against CCl4-induced neurotoxicity in Wistar rats.
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Materials and methods
Sample preparation
Chemicals
Post-mitochondrial supernatant (PMS) and homogenate preparation
Sulfosalicylic acid and thiobarbituric acid (TBA) were obtained from Hi-Media Labs (Mumbai, India). Butylated hydroxytoluene (BHT), o-phosphoric acid (OPA), trichloroacetic acid (TCA), 1-Amino-2-naphthol-4-sulfonic acid (ANSA), 2,4-dinitrophenylhydrazine (DNPH), and perchloric acid (PCA) were purchased from Merck Limited (Mumbai, India). Bovine serum albumin, reduced glutathione (GSH), oxidized glutathione (GSSG), and benzylamine hydrochloride (BAHC) were purchased from Sigma Chemicals Co. (St. Louis, MO). Reduced nicotinamide adenine dinucleotide phosphate (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), ethylenediaminetetraacetic acid (EDTA), and acetylthiocholine iodide (ATC) were obtained from SRL (Mumbai, India). Other routine chemicals were obtained from Hi-Media Labs (Mumbai, India), Merck Limited (Mumbai, India), and SRL (Mumbai, India). CCl4 and HP were obtained from Merck Limited (Mumbai, India) and SRL Labs (Mumbai, India), respectively. Animals Adult male Wistar rats weighing 250–300 g were used for the study. The experimental rats were acclimatized for a week prior to the commencement of the dose regime. The animals were housed under standard laboratory conditions where temperature was maintained at 22 ±2 C with a relative humidity of 65 ± 10% and a photoperiod of 12 h light/dark cycle. All the experiments were carried out according to the standard guidelines of Institutional Animal Ethics Committee (IAEC) of Jamia Hamdard (Hamdard University).
The brain of the animals was promptly excised and washed with chilled phosphate-buffered saline for the removal of blood. The brain was homogenized 1:10 w/v in 0.1 M sodium phosphate buffer (pH 7.4) with a Potter–Elvehjem homogenizer. The homogenates obtained were used to estimate brain TBARS. The homogenates were centrifuged at 10 500 g for 20 min at 4 C to get the PMS, using a REMI C-24 centrifuge (Remi Sales and Engineering, Ltd., Mumbai, India). The PMS supernatant obtained was used for the analysis of oxidative stress biomarkers, non-enzymatic antioxidants, enzymatic antioxidants, and neurotoxicity biomarkers. Biochemical estimation Oxidative stress biomarkers Determination of LPO. The extent of LPO was measured
using the procedure of Tabassum et al. (2007). The concentration of thiobarbituric acid reactive substances (TBARS) was determined using an extinction coefficient of 1.56 105 M1 cm1 and results were expressed as nmol TBARS formed/h/mg protein. Protein oxidation in brain PMS was measured as a concentration of protein carbonyls (PC) formed. PC level was determined using DNPH assay according to the procedure of Chaudhary & Parvez (2012). The results were expressed as nmol DNPH incorporated/mg protein based on the molar extinction coefficient of 2.1 104 M1 cm1.
Determination of protein oxidation.
Non-enzymatic antioxidants Experimental design
Estimation of non-protein thiol (NP-SH). The NP-SH was
The animals were randomly divided into four groups, and each group consisted of seven animals. The description about the different groups is as follows: Group 1: Control group. The animals were treated with a vehicle for HP (i.e. normal saline) by oral route for eight consecutive days and a single subcutaneous (s.c.) injection of olive oil on the 7th day. Group 2: HP group. In this group, rats received HP (200 mg/ kg b.w., oral) treatment for a period of 8 d. Group 3: CCl4 group. The animals were administered normal saline for 8 d and were exposed to CCl4 (2 ml/kg b.w., s.c., 40% v/v in olive oil) on the 7th day of the treatment. Group 4: HP (200 mg/kg b.w., oral) + CCl4 (2 ml/kg b.w., s.c.). Rats were given HP treatment for 8 d and a single injection of CCl4 on the 7th day along with HP. The dose of CCl4 and HP used in our study was based on previously published research reports (El-Sayed et al., 2008; Jayakumar et al., 2008). At the end of the experimental period of 8 d, the animals were anesthetized and sacrificed by decapitation. The brain was immediately dissected, and washed in chilled phosphate buffer to remove blood. The effect of CCl4 and role of HP on the oxidative stress biomarkers were studied in the brain tissue.
measured according to the method described by Govil et al. (2012). The molar extinction coefficient of 13 100M1 cm1 at 412 nm was used for the determination of the NP-SH content. The values were expressed as nmol of the NP-SH/mg protein. Estimation of reduced glutathione (GSH). The GSH content
was estimated according to the method of Chaudhary & Parvez (2012). The GSH concentration was calculated as nmol GSH/mg protein using a molar extinction coefficient of 1.36 104 M1 cm1. Enzymatic antioxidants The activity of GPx was assayed according to the method of Haque et al. (2003). The assay mixture consisted of sodium phosphate buffer (0.1 M, pH 7.4), EDTA (1 mM), sodium azide (1 mM), GSH (1 mM), NADPH (0.02 mM), H2O2 (0.25 mM), and 0.1 ml of PMS of brain in a total volume of 2 ml. Oxidation of NADPH was recorded kinetically at 340 nm. The enzyme activity was calculated as nmol NADPH oxidized/min/mg protein, using a molar extinction coefficient of 6.22 103 M1 cm1.
Activity of glutathione peroxidase (GPx).
The activity of GR was assayed by the method of Haque et al. (2003). The assay
Activity of glutathione reductase (GR).
Hesperidin as natural antioxidant
DOI: 10.3109/15376516.2014.945108
system consisted 0.1 M phosphate buffer (pH 7.4), 0.5 mM EDTA, 1 mM GSSG, 0.1 mM NADPH, and 0.3 ml supernatant of brain tissue in a total volume of 2 ml. The enzyme activity was quantitated at 25 C by measuring the disappearance of NADPH at 340 nm, and was calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.223 103 M1 cm1.
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homogenate when compared with the control group (Figure 1A). HP (200 mg/kg, oral) pre-treatment revealed a significant (p50.01) depletion in the LPO content of brain homogenate as compared with the CCl4-treated group. HP alone did not show any significant change in the LPO content of animals beyond control values. Effect on protein oxidation (PC)
The activity of GST was assayed using the method of Haque et al. (2003) by the catalytic reaction of CDNB with the sulfhydryl group of glutathione and was measured kinetically at 340 nm. The assay system consisted of 0.1 M phosphate buffer (pH 7.4), 10 mM GSH, 10 mM CDNB, and 0.1 ml of PMS of rat brain. The enzymatic activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar coefficient of 9.6 103 M1 cm1.
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Activity of glutathione-s-transferase (GST).
Exposure to CCl4 resulted in a significant (p50.001) enhancement in the activity of protein oxidation in the brain PMS of CCl4-treated animals as compared with the control group (Figure 1B). In HP pre-treated group, there was a significant (p50.01) depletion in the PC content of brain PMS. However, no significant effect in the PC content of HP treated group was observed when compared with control. Non-enzymatic antioxidant status
Neurotoxicity biomarkers
Effect on NP-SH
Activity of monoamine oxidase (MAO). The activity of MAO
The level of NP-SH in brain PMS significantly (p50.001) increased in CCl4-administered group of animals when compared with the animals of the control group (Figure 2A). CCl4-exposed HP pre-administered animals showed a significant (p50.001) decrease in the NP-SH level as compared with that of CCl4-administered animals.
was measured by using the method of Chaudhary & Parvez (2012). The enzyme activity was calculated as mmol BAHC hydrolyzed/min/mg protein using a molar extinction coefficient of 7.6925 M1 cm1. Activity of acetylcholinesterase (AChE). The activity of AChE
was estimated by using the method of Chaudhary & Parvez (2012). The enzyme activity was calculated as nmol ATC hydrolyzed/min/mg protein using a molar extinction coefficient of 1.36 104 M1 cm1. The activity of total ATPase was measured as the release of inorganic phosphate (Pi) by the method of Chaudhary & Parvez (2012). The activity was measured as mg Pi liberated/min/mg protein.
Activity of total ATPase.
Determination of protein Estimation of protein contents in biological fractions of brain was done using bovine serum albumin as standard by the method of Lowry et al. (1951). Statistical analysis The results obtained were expressed as mean ± standard error (SE). All data were analyzed using analysis of variance (ANOVA) followed by Tukey’s test. Values of p50.05 were considered as significant. All the statistical analyses were performed using graph pad prism 5 software (Graph Pad Software, Inc., San Diego, CA).
Results No mortality and significant alteration in the body weight of different groups of subject/animals were documented during and after the treatment (data not shown). Oxidative stress biomarkers Effect on LPO Treatment of animals with CCl4 (2 ml/kg b.w.) showed a significant (p50.001) elevation in the LPO content of brain
Figure 1. Effect of CCl4 and HP on (A) LPO and (B) PC in brain of rat. Values were expressed as nmoles of TBARS formed/h/mg protein and nmoles of DNPH incorporated/mg protein, respectively. Each value represented as mean ± SE (n ¼ 6). Significant differences were indicated by ***p50.001 when compared with control and significant difference were indicated by ##p50.01 when compared with CCl4-treated animals.
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CCl4 significantly increased (p50.001) the glutathione reductase level (GR) in CCl4-treated group as compared with the control group (Figure 3B). A single dose of CCl4 combined with pretreatment of HP showed a significant (p50.01) decrease in GR activity as compared with the CCl4intoxicated group. HP treatment showed no significant change in the activity of GR in the PMS of brain. CCl4 exposure showed a significant (p50.001) rise in the activity of GST when compared with the control group (Figure 3C). In a single dose of CCl4 in the HP pre-treated group revealed a significant (p50.001) decline in the GST activity in brain PMS as compared with the CCl4-treated group. Group treated with HP showed no effect in the activity of GST. Effect on neurotoxicity biomarkers
Figure 2. Effect of CCl4 and HP on level of (A) NP-SH and (B) GSH in brain tissue of rat. Values were expressed as nmoles NP-SH/mg protein and nmoles GSH/mg protein, respectively. Each value represented as mean ± SE (n ¼ 6). Significant differences were indicated by ***p50.001 when compared with control. Significant differences ###p50.001 were shown when compared with CCl4-treated animals.
HP-treated animals showed no any significant alteration in the NP-SH level in brain PMS when compared with control group of animals. Effect on GSH Significant (p50.001) higher level of the GSH content was observed in brain PMS of CCl4-exposed animals when compared with the control group (Figure 2B). HP pretreatment prior to CCl4 injection significantly (p50.001) declines the GSH content and prevented these elevations. Animals treated with HP did not show any significant change when compared with the control group. Enzymatic antioxidants activities Effect on glutathione-metabolizing enzymes There was a significant (p50.001) elevation in the activity of GPx in brain PMS in the CCl4-treated group as compared with the control group (Figure 3A). HP pretreatment showed a significant (p50.001) decline in the activity of GPx as compared with the CCl4-treated group. HP-treated group did not change the GPx level in the brain PMS when compared with the control group.
There was a significant (p50.001) increase in the activity of MAO in the CCl4-treated group of animals (Figure 4A). HP supplementation along with a single dose of CCl4 showed a significant (p50.001) decline in the activity of MAO as compared with that of the CCl4-treated group. No effect in the activity of MAO was seen in the HP-treated group. The CCl4-treated group showed significant (p50.001) rise in the activity of AChE as compared with the control group (Figure 4B). The HP pre-exposed group significantly (p50.001) decreased the activity of AChE when compared with the CCl4-treated group and prevented such elevations. The HP-treated group exhibited no remarkable difference when compared with control. A single dose of CCl4 caused a significant (p50.01) increase in the total ATPase content in brain PMS of animals when compared with control (Figure 4C). HP pre-treatment in CCl4-treated group caused significant (p50.01) decline in the activity of ATPase. The HP-treated group did not show significant change in the activity.
Discussion Rats intoxicated with CCl4 leads to the generation of ROS, ultimately resulting in neurotoxicity in rats (Samudram et al., 2009). These highly reactive free radicals of CCl4 generated by the CYP are capable of covalently binding to macromolecules and cause intense LPO leading to the degeneration of cell membrane of endoplasmic reticulum which is eminently rich in PUFA (Altas et al., 2011). HP possesses highest reducing power and exhibits anti-oxidative properties (Salem et al., 2012). TBARS is a by-product of LPO, considered as a reliable index of tissue damage (Tabassum et al., 2007). In the present study, increased levels of TBARS in brain tissue may be due to increased production of these free radicals which are unstable and highly active, leading to fatty acid damage following CCl4 intoxication which will lead to enhanced LPO. The potential amelioration of free radicals which leads to fatty acid damage by HP, which is a wellknown antioxidant reducing the severity of LPO by decreasing TBARS concentration. It suggested that HP exerts its protective effect by regulating LPO of membranes by scavenging the debilitating consequences of free radicals and augmenting the antioxidant defence system (Pradeep et al., 2008).
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DOI: 10.3109/15376516.2014.945108
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Figure 3. Effect of CCl4 and HP on (A) GPx, (B) GR and (C) GST activity in brain of rat. GPx and GR activities were expressed as nmoles of NADPH oxidized/min/mg protein whereas GST activity was expressed as nmoles of CDNB conjugate formed/min/mg protein. Each value represented as mean ± SE (n ¼ 6). Significant differences were shown by ***p50.001 when compared with control. Significant differences were indicated by ##p50.01 and ###p50.001 when compared with CCl4-treated animals.
PC is considered as an excellent biomarker of oxidative protein damage as proteins are susceptible to oxidative modification (Dalle-Donne et al., 2003). Oxidative modification of proteins changes its configuration via aggregation, fragmentation, and formation of cross linkages in the polypeptide chain and also leads to secondary impairment of biomolecules with loss of protein biological function, leads to the generation of superoxide radical (Chen et al., 2005). Addition of carbonyl group in proteins is directly proportional to the protein subjected to oxidative damage and an increase in carbonyl content is associated with various pathological disorders (Almroth et al., 2005). A significant increase in protein oxidation was seen in the brain of animals with the single-dose administration of CCl4 as compared with the control group. It might be due to increased oxidatively modified protein in brain with the administration of CCl4. Our result is in agreement with the findings of previous result performed with liver (Srivastava & Shivanandappa, 2010). Interestingly, pre-administration of HP attenuated the effect of CCl4-induced protein damage, indicating that HP has a protective action against CCl4-induced neurotoxicity. Our results are in corroboration with the recent finding where HP has been shown to be a good natural antioxidant against cisplatin-induced oxidative damage (Sahu et al., 2013). HP has been found to reduce the PC content in our study thereby reducing oxidative stress and oxidative damage of protein by
scavenging the free radicals causing modification in the protein structure. Dietary antioxidant supplementation such as bioflavonoids mainly protects cells and organs against ROS-induced oxidative damage (Tsai et al., 2013). GSH scavenges free radicals and provides a defence system against ROS as well as detoxifies various xenobiotic compounds, H2O2, lipid hydroperoxides, and electrophilic compounds (Manna et al., 2011). It plays crucial role in defence mechanism and also acts as a co-enzyme for numerous enzymes involved in the cellular defence system (Masella et al., 2005). GPx constitutes a family of enzyme which reduces H2O2 and lipid hydroperoxides to the corresponding stable hydroxy compounds and H2O utilizing GSH as a reducing substrate (Dickinson & Forman, 2002). GR is a flavoenzyme and is represented by a single copy gene in humans which takes part in the reduction of GSSG (oxidized glutathione) to GSH with the help of NADPH (Li et al., 2010). The activity of GR is very important to maintain the level of GSH/GSSG ratio. GST is a well-known phase-2 enzyme which plays a very crucial role in the detoxification process of electrophilic compounds, favoring the elimination from the body of the organ by conjugation with glutathione (Afzal et al., 2013). GSH oxidizes to GSSG by peroxide and GPx. GSH is restored from GSSG by the enzyme GR using NADPH as an electron donor and the ratio of GSH/GSSG within a cell is considered as the measure of
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Figure 4. Effect of CCl4 and HP on (A) MAO, (B) AChE, and (C) total ATPase activity in brain of rat. MAO activity was expressed as mmol BAHC hydrolyzed/min/mg protein, AChE activity as nmol ATC hydrolyzed/min/mg protein and total ATPase activity as mg Pi liberated/min/mg protein. Each value represented as mean ± SE (n ¼ 6). Significant differences were shown by **p50.01 and ***p50.001 when compared with control. Significant differences were indicated by ##p50.01 and ###p50.001 when compared with CCl4-treated animals.
cellular toxicity (Board & Menon, 2013). An increase in the activity of GR, GPx, and GST was found in the CCl4intoxicated group of animals. An increase in the activity of glutathione-dependent enzymes reveals the primary response of the cell to the exposure to oxidative stress-inducing toxic compounds with the potential of inducing oxidative stress and damage caused in the cells and the cells may reduce the severity of oxidative damage by maintaining a reduced state in the cell. Induction in the activity of GPx on CCl4 administration suggests that there might be a rapid oxidation of GSH mediated by peroxide. This result is in agreement with the findings of the effect of cadmium (Cd) on different organs, liver, kidney, and gills of freshwater murrel Channa punctatus (Dabas et al., 2012). The increased level of GST in CCl4intoxicated animals suggests that it might be due to the conjugation between trichloromethyl radical and GSH to mitigate the effect of increased oxidative stress. Our result is in agreement with the result obtained earlier in the brain of the CCl4-treated group of animals (Soliman & Fahmy, 2011). HP administration restored the increased level of GSH which suggests the reversal of the increased oxidative stress. Our result corroborates with the findings of antioxidative and antiinflammatory role of HP on the rat air pouch model of inflammation (Jain & Parmar, 2011). It may be due to the depletion in the level of cysteine with the pretreatment of HP as cysteine which is one of the major constituents of tripeptide
antioxidant GSH which may affect the level of GSH in the HP pre-treated CCl4-intoxicated group of animals. MAO, AchE, and Na+/K+-ATPase are considered as important neurotoxicity biomarkers. MAO is a flavincontaining brain-specific enzyme which plays an important role in oxidative deamination of endogenous monoamine neurotransmitters, regulates the oxidative deamination of endogenous and exogenous monoamines in the central nervous system, and converts biogenic amines to their corresponding aldehydes (Secci et al., 2011). Alteration in the function of MAO may lead to age-related disorder and a number of neurological disorders and depression (Meyer et al., 2006). MAO takes part in the metabolism of catecholamine like adrenaline, noradrenaline, and dopamine into free radicals, abundance of which may be responsible for serotonin syndrome (Ghareeb et al., 2011). An increase in the activity of MAO suggests that there is a high level of oxidative stress. In the present assessment, a single dose of CCl4 was found to be effective in increasing the activity of MAO. Pretreatment of animals with HP inhibited the activity of MAO. HP was found to be an effective inhibitor of MAO as it inhibited the metabolism of the monoamines metabolization of which form hydrogen peroxide and ammonia which is the potential toxic products of MAO. AchE is a key enzyme in the nervous system which is responsible for the degradation of neurotransmitter,
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DOI: 10.3109/15376516.2014.945108
acetylcholine (Ach), during neurotransmission which is necessary for learning and memory deficits (Parent & Baxter, 2004) and also plays a very important role in the cholinergic activity (Antoniades et al., 2002). Our result is in accordance with a recent finding in which an increase in the activity of AchE in the region of brain has been reported with streptozotocin treatment (Biasibetti et al., 2013). An increase in the activity of AchE in the brain is responsible for the low availability of acetylcholine and, therefore, it may lead to the alteration in cholinergic transmission (Tiwari et al., 2009). HP pre-treatment was able to reverse the altered activity of AchE which deals with the cognitive defect by increasing cholinergic neurotransmission as reported in a report with a natural antioxidant curcumin (Agrawal et al., 2010). The sodium potassium adenosine triphosphate (Na+/K+ATPase) is an enzyme which maintains neuronal excitability and plays a major role in the export and import of three Na+ ions out and two K+ ions in of the cell by utilizing ATP across the plasma membrane against their potential gradient in the nervous system and Na+/K+-ATPase has also been reported as a signal transducer (Lingrel, 2010). The Na+/K+-ATPase is an integral membrane heterodimer belongs to a superfamily of proteins collectively designated as P-type ATPase family (Jaitovich & Bertorello, 2006). In the present study, a remarkable elevation is shown in the activity of Na+/K+ATPase in the CCl4-intoxicated group of rats. HP pretreatment suppressed the elevated level of Na+/K+-ATPase activity in brain due to its neuroprotective nature. Our result is in agreement with the previous result reported with some natural compounds, green tea and bamboo leaves on Na efflux channel in ovariectomized rats (Ryou et al., 2012). Further studies are required for deciphering cellular and molecular mechanisms of experimentally induced neurotoxicity and the role of HP in its attenuation.
Declaration of interest The authors declare that they have no conflict of interest. University Grants Commission (UGC), Government of India, is gratefully acknowledged for providing funding under Faculty Research Award to S.P. M.N. was supported by a Junior Research fellowship from UGC – Maulana Azad National Fellowship program.
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