NeuroToxicology 44 (2014) 194–203

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NeuroToxicology

Plant toxin abrin induced oxidative stress mediated neurodegenerative changes in mice A.S.B. Bhasker a,*, Bhavana Sant a, Preeti Yadav a, Mona Agrawal a, P.V. Lakshmana Rao b a b

Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India DRDO-BU Center for Life Sciences, Bharathiar University, Coimbatore 641046, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 February 2014 Accepted 30 June 2014 Available online 7 July 2014

Abrin is a potent plant toxin. It is a heterodimeric protein toxin which is obtained from the seeds of Abrus precatorius plant. At cellular level abrin causes protein synthesis inhibition by removing the specific adenine residue (A4324) from the 28s rRNA of the 60S – ribosomal subunit. In the present study we investigated the role of oxidative stress in neurotoxic potential and demyelinating effects of abrin on brain. The mechanism by which abrin induces oxidative damage and toxicity in brain are relatively unknown. Animals were exposed to 0.4 and 1.0 LD50 abrin dose by intraperitoneal route and observed for 1 and 3 day post-toxin exposure. Oxidative stress occurred in brain due to abrin was confirmed in terms of increased reactive oxygen species (ROS), glutathione depletion and increased lipid peroxidation. Significant increase in blood and brain ROS was observed at day 3, 1 LD50. Abrin induced changes in the neurotransmitters (5-hydroxy tryptamine, norepinephrine, dopamine and monoamine oxidase) levels were evaluated by spectroflourometry. Increase in the levels of 5-HT and NE was observed after abrin exposure. MAO activity was found to be decreased in abrin exposed animals compared to control. Significant inhibition in the activity of acetylcholine esterase enzyme in brain and serum was reported for both the doses and time points. Western blot analysis of iNOS expression indicated that abrin treatment resulted in dose and time dependent increase. Furthermore, protein expression of myelin basic protein (MBP) was down regulated in a dose and time dependent manner. Brain histopathology was carried out and cortical brain region showed demyelination after abrin exposure. Results confirmed that abrin poisoning leads to neurodegeneration and neurotoxicity mediated through oxidative stress, AChE inhibition, lipid peroxidation and decrease in MBP levels. ß 2014 Elsevier Inc. All rights reserved.

Keywords: Abrin Neurotransmitters Acetyl cholinesterase (AChE) Myelin basic protein (MBP) Ribosome inactivating proteins (RIP)

1. Introduction Jequirity bean (Abrus precatorius L) and castor bean (Ricinus communis L.) are known for their medicinal use and toxicity for more than a century (Olsnes, 2004; Franz, 1988). Toxicity of these seeds had been implicated to abrin and ricin, two proteinacious toxins produced by these plants respectively. Molecular target for these toxins is mammalian ribosome, hence known as ribosome inactivating proteins (RIP). Abrin and ricin are classified as type 2 RIPs. They contain an enzymatically active 30 kDa A chain, linked to a slightly larger B chain with a size of 35 kDa. B chain has properties of lectin with specificity for sugars with the galactose structures (Stirpe, 2004; Lord et al., 1994). These toxins act as glycosidases that specifically cleave nucleoside N-glycosidic

* Corresponding author. Tel.: +91 751 2233495; fax: +91 751 2341148. E-mail address: [email protected] (A.S.B. Bhasker). http://dx.doi.org/10.1016/j.neuro.2014.06.015 0161-813X/ß 2014 Elsevier Inc. All rights reserved.

bonds. It has been proposed that RIPs inhibit protein synthesis by virtue of their enzymatic activity, selectively removing a specific adenine residue from the highly conserved and surface exposed alpha sarcin/ricin loop at position 4324 on the conserved GAGA loop of rat liver 28S rRNA. This enzymatic cleavage prevents the binding of the elongation factor, EF-2-GTP complex to the ribosome that subsequently arrest protein synthesis and eventually leads to cell death (Park et al., 2004). At the cellular level both abrin and ricin cause apoptosis and subsequently at higher doses, severe necrosis in the organs of poisoned animals and in cultured cells (Griffiths et al., 1987; Hughes et al., 1996; Narayanan et al., 2004). Ricin showed hepatotoxicity and nephrotoxicity in mice showing oxidative damage potential of the toxin (Kumar et al., 2003). We have earlier reported the oxidative stress mediated DNA damage and cell death by ricin and abrin in vitro (Bhaskar et al., 2008; Rao et al., 2005). One of the important features of in vivo toxicity of ricin and abrin is the vascular leak syndrome. Endothelial cell damage

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leading to tissue edema matches correctly with many observations recorded in abrin poisoning (Dickers et al., 2003). There have been reports of neurological observations during abrin poisoning episodes (Deshpande et al., 1961; Frohne et al., 1984). A 2007 case report describes death of a woman after consuming the Jequirity seeds. The patient died in three days with progressive central nervous system depression. The report describes immune mediated demyelinating encephalitis (Sahni et al., 2007). Demyelination was again reported in another case of suicidal attempt, with the help of a magnetic resonance imaging (MRI) brain scan showing hyper-intensities in the bilateral medial temporal lobes in fluid attenuation inversion recovery (FLAIR), suggestive of demyelination (Sahoo et al., 2008). Ricin and abrin are one of the most toxic substances produced by plants. They are potential candidate toxins for use as a chemical weapon mainly due to its simple extraction procedure and high toxicity in aerosolized form. Sporadic incidents involving ricin reported from many countries with doubtful authenticity of possession (Dickers et al., 2003). Oxidative stress plays a crucial role in the pathogenesis of a large number of neurological diseases and psychiatric disorders, such as Alzheimer’s disease (Behl, 1997), Parkinson’s disease (Dias et al., 2013), Schizophrenia (Boskovic et al., 2011) or Bipolar disorder, Multiple Sclerosis (Ortiz et al., 2013) and also stroke. Oxidative stress mainly occurs due to the excessive production of free radical or low antioxidant defense, and results in chemical alterations of biomolecules causing structural and functional changes (Chiarugi et al., 2003). Neurons are highly susceptible to oxidative stress which may induce both neuronal necrosis and apoptosis. When the generation of oxidants exceeds the rate at which endogenous antioxidant defences can scavenge oxidants, proteins, lipids, DNA and other macromolecules become targets for oxidative modification, which leads to alteration of cellular structure architecture and activation of various signaling pathways and finally death (Ischiropoulos and Beckman, 2003). In the present study we investigated the abrin induced oxidative damage in brain. Alterations in neurotransmitter levels and markers of demyelination in brain were also studied. Results of our study clearly show that at sub-lethal dose also abrin can cause neurodegenerative changes.

2. Materials and methods 2.1. Chemicals Abrin was isolated from A. precatorius seeds as described by Kumar et al. (2008). The purified abrin was lyophilized and stored at 80 8C and reconstituted as and when required in PBS. Reduced glutathione (GSH) was from Across (Belgium). O-phthaldialdehyde (OPT) was from Fluka (USA). MBP and iNOS antibodies from Cell signaling (India). Anti-b-actin (Clone AC-74) was obtained from Sigma (USA). Anti-mouse and anti-rabbit HRP conjugated antibody was obtained from DAKO (Denmark). All other chemicals were obtained from Sigma chemical Co. (St. Louis, USA) unless otherwise mentioned. 2.2. Animals Randomly bred Swiss albino male mice weighing between 24 and 28 g body mass from Establishment’s animal facility were used for the study. The animals were housed in polypropylene cages with dust-free, sterile rice husk as bedding material, and were provided with pellet food (Ashirwad Industries, Chandigarh, India) and water ad libitum.

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2.3. Experimental design Animals were divided into five groups (Group 1 – control, Group 2 – 0.4 LD50 1 day, Group 3 – 0.4 LD50 3 day, Group 4 – 1.0 LD50 1 day and Group 5 – 1.0 LD50 3 day) of 9 animals per group. In each group, 6 animals were used for biochemical assays and 3 animals for histopathology study. Animals were allowed to acclimatize 7 days prior to dosing. Abrin toxin was diluted in PBS before animal exposure. Mice were administered 0.4 LD50 (1.13 mg/kg) and 1.0 LD50 (2.83 mg/kg) of abrin toxin by intraperitoneal route of exposure. The doses were chosen with a difference of 2.5 folds. Control mice received equal volume PBS by same route. The care and maintenance of the animals were as per the approved guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. This study has the approval of Institute’s ethical committee on animal experimentation. 2.4. Biochemical assays Body weight and food intake of animals was monitored daily till 3 days post-exposure. For biochemical studies, blood was drawn from orbital plexus before sacrificing the animals. Soon after blood sampling, animals were sacrificed by cervical dislocation, brain was excised quickly. Brain tissue was washed free of adhering extraneous material and blotted. Blood and brain ROS was estimated fluorimetrically (Socci et al., 1999). Brain GSH content was estimated spectrofluorimetrically (Hissin and Hilf, 1976). Brain lipid peroxidation was measured following the procedure of Ohkawa et al. (1979). 2.5. Brain and serum acetyl cholinesterase (AChE) A 10% brain homogenate (w/v) was prepared in 0.25 M sucrose. Activity of acetyl cholinesterase (AChE) in brain and serum was determined according to the method of Ellman et al. (1961) using acetylthiocholine as substrate. The activity of AChE was measured at 412 nm and its unit is expressed as nmol/min/mg protein. 2.6. Brain monoamine oxidase (MAO) MAO activity was studied in brain mitochondrial fraction following the method of Wurtman and Axelrod (1963). Briefly, 1.0 ml of 0.2 M phosphate buffer (pH 7.2) and 0.8 ml distilled water was added to 100 ml of mitochondrial fraction. 0.1 ml of benzylamine HCl (0.1 M, pH 7.2) was added to the abrin treated samples. The tubes were incubated for 30 min at 37 8C and the reaction was stopped by adding 1 ml of 10% perchloric acid. For control samples, instead of mitochondrial fraction equal amount of phosphate buffer was added. After centrifugation, the supernatant was diluted with equal volume of distilled water and read at 250 nm. The enzyme activity was expressed as nM of benzaldehyde formed/min/mg protein. 2.7. Brain biogenic amines The frozen brain tissue samples were weighed and homogenized in acidified butanol. Dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT) were estimated according to the procedure of Jacobowitz and Richardson (1978). Briefly, aliquots of butanol extracts were re-extracted with phosphate buffer (0.1 M, pH 6.5), part of which was derivatized by adding sodium EDTA and iodine solution. The reaction was terminated by alkaline sulphite and neutralized with acetic acid (5 N). Fluorescence was read by excitation at 385 nm and emission at 485 nm for NE, and 320 nm and 385 nm respectively, for DA. To determine 5-HT, the butanol

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layer was extracted with 0.1 N HCl in the presence of n-heptane and the acid layer was mixed with o-phthaldehyde. After boiling and cooling, fluorescence was read by excitation at 360 nm and emission at 470 nm. 2.8. Western blot analysis Brain tissue samples were processed for SDS-PAGE followed by Western blot. The brain tissues from control and various treatment samples were homogenized with 5–10 volumes of lysis buffer (10 mM HEPES pH 7.4, 42 mM KCl, 50 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 5 mM DTT, 2 mM PMSF, 1 complete protease inhibitor cocktail). Cellular debris were spun down at 10,000  g for 20 min and supernatants were used as whole protein extract, aliquoted and stored at 80 8C till processing. The total protein concentration was estimated using Bio-Rad method. Thirty micrograms of protein from each sample was separated on SDSPAGE and electrophoretically transferred on to a nitrocellulose membrane, using an electro-blotting apparatus. Membranes were incubated in blocking solution containing 5% non-fat dry milk in TBST buffer (TBS buffer containing 0.1% Tween-20) for 1 h at room temperature, followed by over night incubation at 4 8C in platform shaker with various primary antibodies at specified dilutions: iNOS (1:2000), MBP(1:1000) and b-actin (1:5000). All antibodies are diluted in TBST with 5% milk powder. The membranes were washed four times in TBST for 5 min each, followed by incubation for 2 h in horseradish peroxidase-conjugated anti-mouse or antirabbit secondary antibody used at 1:50,000 dilutions. The membranes were washed again and developed using an enhanced chemiluminescent detection system (ProteoQwestTM Chemiluminescent Western blotting kit, Sigma) according to manufacturer’s protocol and the image was taken on Pierce CL-XPosureTM X-ray films.

3.2. Effect on reactive oxygen species (ROS) Single intraperitoneal injection of abrin induced oxidative stress in blood and brain. Dose and time dependent increase in blood and brain ROS levels was observed in abrin exposed groups as compared to control. Increase in blood ROS at 0.4 LD50, 1 and 3 day post toxin exposure were measured as 0.068  0.00046 and 0.0695  0.00092 nmol of DCF formed/min/ml blood respectively. Similarly, 0.072  0.00489 and 0.076  0.0062 nmol of DCF formed/ min/ml blood were measured for 1.0 LD50, 1 and 3 day respectively (Fig. 1A). Significant increase in blood ROS was observed at 1.0 LD50, 3 day. Similarly, increase in brain ROS at 0.4 LD50, 1 and 3 day post toxin exposure were recorded as 0.0975  0.00745 and 0.112  0.004 nmol of DCF formed/min/mg protein. At 1.0 LD50, 1 and 3 day brain ROS levels were recorded as 0.099  0.0009 and 0.119  0.012 nmol of DCF formed/min/mg protein (Fig. 1B). Significant increase in brain ROS levels was recorded at 3rd day, 1 LD50 dose. Control values for ROS generation in blood and brain were found to be 0.059  0.0045 nmol of DCF formed/min/ml of blood and 0.087  0.0024 nmol of DCF formed/min/mg protein in brain. 3.3. Effect on reduced glutathione and lipid peroxidation (GSH and TBARS) Intracellular GSH levels were measured in brain of abrin exposed mice. Significant depletion was observed compared to control only on day 3 at 0.4 LD50 and on both days of observation in A.

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2.9. Brain histopathological analysis Control and abrin treated animals were sacrificed after specific time points. Brain was dissected out and fixed in Bouin’s solution. After fixation, small pieces were processed by automated tissue processor (Leica TP1020) dehydrated and embedded in paraffin wax. Multiple sections of 12-mm thickness were prepared using automatic microtome (Microm HM360) and stained with hematoxylin and eosin in Leica Autostain-XL. Microscopic observation was performed under LEICA DMLB microscope and photographs were taken using Leica DC 500 camera.

3. Results 3.1. Abrin toxicity In the present study animals were exposed to 0.4 and 1.0 LD50 abrin dose and observed for 1 and 3 day post toxin exposure. Abrin exposed animals showed low food and water intake, weight loss and severe diarrhea in time and dose dependent manner. No mortality was observed in 0.4 LD50 abrin exposed animals. 1.0 LD50 abrin exposed animals showed mortality from day 2 onwards and 50% animals died by day 3.

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Fig. 1. Effect of 0.4 (1.132 mg/kg) and 1 LD50 (2.83 mg/kg) of abrin exposure by intraperitoneal route on reactive oxygen species (ROS) after 1 and 3 days postexposure. (A) Blood and (B) Brain. Values are mean  SE of six mice per group. * Signficantly different from control group and means followed by different alphabet(s) in abrin exposed groups are significantly different at P  0.05 by Student Newman Keuls multiple comparison test.

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1.0 LD50 dose group. GSH depletion was dose and time dependent with maximum effect observed in 3 day, 1.0 LD50 group. GSH in control group was found to be 24.05  0.44 mg GSH/g tissue and in exposed groups at 0.4 LD50, 1 and 3 day (22.74  2.3 and 18.83  0.6) and at 1.0 LD50, 1 and 3 day (18.4  0.32 and 17.27  0.45) mg GSH/g tissue respectively (Fig. 2A). Brain lipid peroxidation was measured quantitatively as thio barbituric acid reactive substances (TBARS). Brain TBARS level significantly increased at 0.4 LD50, both days and at 1.0 LD50 3 day. Control levels of TBARS were 7.35  0.31 mg/g of tissue and in abrin exposed groups at dose 0.4 LD50, 1 and 3 day TBARS levels were noted as 8.5  0.29 and 8.68  0.095 respectively and at 1.0 LD50, 1 and 3 day were 8.02  0.153 and 8.48  0.377 mg/g of tissue respectively (Fig. 2B). 3.4. Effect on acetyl cholinesterase activity Exposure to abrin showed a significant inhibition of brain and serum AChE activity in all the groups. Maximum AChE inhibition was observed at 1 LD50 3 day post exposure (Fig. 3A and B). Dose and time dependent decrease in brain and serum AChE activity was seen. In brain tissue 0.035  0.0052 (70.40%) and 0.031  0.0021 (62.29%) nmol/min/mg protein AChE activity was recorded at the dose of 0.4 LD50 for 1 and 3 days, respectively. While 0.032  0.0043 (62.49%) and 0.0292  0.0022 (58.42%) nmol/min/mg protein, brain activity was remaining at 1.0 LD50 abrin exposure on 1 and 3 days showing dose dependent effect. Control brain AChE activity was A.

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recorded as 0.050  0.0017, which is considered as 100% brain AChE activity. Serum AChE activity at the dose of 0.4 LD50 was 0.65  0.037 (65.12%) and 0.41  0.037 (41.55%) on 1 and 3 days respectively, while at 1.0 LD50 the enzyme activity was 0.45  0.073 (45.38%) and 0.36  0.031 (36.68%) nmol/min/mg protein on 1 and 3 days respectively. Control serum AChE activity was 1.00  0.025 nmol/ min/mg protein and considered as 100% serum AChE activity. 3.5. Effect on brain MAO and biogenic amines Brain mono amine oxidase activity in mice inhibited significantly in all treatment groups. Inhibition was dose dependent. 5HT and NE registered a similar trend of elevation in brain under acute exposures of abrin toxin against the control. Increase in 5-HT at 0.4 LD50 and 1.0 LD50 dose was recorded for both the time points. Significant increase in 5-HT levels was measured at 1 day, 1 LD50 dose. Control levels of 5-HT was recorded as 107.03  1.91 ng/g of tissue (Fig. 4A). Significant depletion in the MAO activity was recorded as 0.165  0.041 and 0.122  0.008 nmol of benzaldehyde formed/min/mg protein at 1 and 3 day of 0.4 LD50 dose. At 1 and 3 day of 1.0 LD50 dose MAO activity was 0.141  0.0073 and 0.1108  0.0054 nmol of benzaldehyde formed/min/mg protein (Fig. 4B). Control levels of MAO observed as 0.214  0.019 nmol of benzaldehyde formed/min/mg protein. Similarly, elevation in NE was also noticed in brain. NE was found to be 125.66  8.38 ng/g of tissue in control mice. NE brain levels was significantly increased at both the doses and time points and recorded as 217.07  7.79 and 205.54  10.85 at 0.4 LD50 and 201.45  6.82 and 181.59  1.08 at

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Fig. 2. Effect of 0.4 (1.132 mg/kg) and 1 LD50 (2.83 mg/kg) of abrin exposure by intraperitoneal route on brain (A) GSH and (B) lipid peroxidation after 1 and 3 days post-exposure. Values are mean  SE of six mice per group. * Signficantly different from control group and means followed by different alphabet(s) in abrin exposed groups are significantly different at P  0.05 by Student Newman Keuls multiple comparison test.

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Fig. 3. Effect of 0.4 (1.132 mg/kg) and 1 LD50 (2.83 mg/kg) of abrin exposure by intraperitoneal route on AChE (A) Serum and (B) Brain after 1 and 3 days postexposure. Values are mean  SE of six mice per group. * Signficantly different from control group and means followed by different alphabet(s) in abrin exposed groups are significantly different at P  0.05 by Student Newman Keuls multiple comparison test.

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Fig. 4. Effect of 0.4 (1.132 mg/kg) and 1 LD50 (2.83 mg/kg) of abrin exposure by intraperitoneal route on brain biogenic amines (A) 5-HT, (B) MAO, (C) NE, and (D) DA after 1 and 3 days post-exposure. Values are mean  SE of six mice per group. * Signficantly different from control group and means followed by different alphabet(s) in abrin exposed groups are significantly different at P  0.05 by Student Newman Keuls multiple comparison test.

1.0 LD50 on 1 and 3 day respectively (Fig. 4C). However, DA levels showed depletion in all treated groups At 3 day of both the doses DA was recorded as 37.5  4.04 and 34.17  5.15 ng/g of tissue respectively. Control levels are 43.96  4.07 ng/g of tissue (Fig. 4D). 3.6. Effect on iNOS and myelin basic protein

region, control mice showed normal granular and purkinje cells layer (Fig. 7A). Cerebellum section of mice brain exposed to 0.4 LD50 of abrin at 1 and 3 day showed pyknosis of purkinje cells with condensed nuclei, increased intracellular space and bright eosinophilic cytoplasm of granular layer indicative of necrosis (Fig. 7B). These changes are more severe at day 3 (Fig. 7C). As

Western blot analysis of iNOS and MBP protein was carried out. Dose and time dependent over expression of iNOS protein was noted in both the doses and time points (Fig. 5A). Myelin basic protein is a key protein in myelin sheath integrity. Protein expression of MBP was found to be down regulated in a dose and time dependent manner (Fig. 5B). 3.7. Effect of abrin on brain histology Cytologic alteration in different regions of brain (cortex, cerebellum and hippocampus) is examined by histopathology. H & E stained sections of control cortex region showed the normal glial cell layer, and pyramidal cells (Fig. 6A). Whereas neuronal degeneration and congestion was observed in brain cortex region after 0.4 LD50 abrin at 1 day post-exposure (Fig. 6B) and at 3 day post-exposure neuronal gliosis and necrotic neurons were observed in brain cortex (Fig. 6C and D). These results are in agreement with MBP activity. 1.0 LD50 abrin treated cortex regions at 1 day and 3 day showed neuronal degeneration and pyknotic neuronal changes (Fig. 6E and F). The classic monomorphic pattern of dark neurons is indicative of degeneration. In contrast, cerebellum is comprised of single layer formed by large sized projection neurons called purkinje layer. In cerebellum

Fig. 5. Immunoblot of (A) iNOS and (B) Myelin basic protein in brain after 0.4 (1.132 mg/kg) and 1 LD50 (2.83 mg/kg) of abrin exposure by intraperitoneal route after 1 and 3 days post-exposure. b-actin was used as protein loading control. Bands pixel density was measured using NIH Image J free software and expressed as fold change in band pixel density over control.

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Fig. 6. Photomicrographs of control, and abrin treated (0.4 and 1 LD50 dose) mice brain Cortex H & E 40. (A) Control mice showing normal glial cells layer, and pyramidal cells. (B) Section of mice brain with Abrin (0.4 LD50, 1 day) showing neuronal degeneration (arrow) and congestion in cortex. (C) Mice treated with Abrin (0.4 LD50, 3 day) showing neuronal gliosis (arrow). (D). 0.4 LD50 3 day abrin treated brain cortex showing necrotic neurons (arrow). (E) and (F) 1 LD50 1 day and 3 day of abrin administered mice brain histology showed neurodegeneration (arrow) and pyknotic (arrow) changes in cortex region.

compared to 0.4 LD50 dose, 1 LD50 dose of abrin, administered mice brain histology at both 1 and 3 day showed increase in pyknotic neurons and necrosis in granular layer (Fig. 7D, E and F). H & E stained hippocampus region of control mice brain, showed normal cellular composition arranged in three layers (Fig. 8A and D). 0.4 LD50 abrin exposed sections of hippocampus at 1 and 3 day showed eosinophilic (degenerating) neurons (Fig. 8B and C). In our results, degenerating neurons are characterized by cell body shrinkage, intensely stained eosinophilic cytoplasm, and a small/ shrunken darkly stained (pyknotic) nucleus that may eventually fragment. 1 LD50 dose of abrin showed time dependent severity of necrosis in pyramidal cells of hippocampus (Fig. 8E and F). 4. Discussion Plant toxic proteins belong to a group of phytotoxins, which inhibit the protein synthesis of eukaryotic cells (Patocˇka and

Strˇeda, 2003). Abrin and ricin are two similar proteins that share significant similarities at the level of sequence as well as the structure. Toxicity wise abrin is many times more potent than ricin. Abrin and ricin toxins are known to cause systemic toxicity with severe intestinal toxicity. Common symptoms of abrin or ricin poisoning are abdominal pain, vomiting and bloody diarrhea. At cellular level, cell death occurs due to protein synthesis inhibition. Previous studies reported vascular leak syndrome as one of the feature of abrin poisoning, in which there is endothelial cell damage, and an increase in capillary permeability with protein leakage and tissue edema (Dickers et al., 2003). Few reports are available on direct toxic effect of abrin on the central nervous system (Deshpande et al., 1961; Frohne et al., 1984), but this has not been substantiated with further studies. Organ toxicity and vascular disturbances in the case of ricin exposure was attributed more to the cell death rather than a direct effect of the toxin itself (Franz et al., 1997; Howat, 1988). CNS related symptoms are now

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Fig. 7. Photomicrographs of control, and abrin treated (0.4 and 1 LD50 dose) mice brain cerebellum H & E 40. (A) Control mice showing normal molecular or granular layer and purkinje layer. (B) Section of mice brain with Abrin (0.4 LD50, 1 day) showing pyknotic purkinje cells (arrow), increased intracellular space and eosinophillic appearance of granular layer is indicative of necrosis. (C) Mice treated with Abrin (0.4 LD50, 3 day) showing more severe changes as compared to day 0.4 LD50, 1 day. (D) 1 LD50 1 day abrin exposed brain cerebellum region showed pyknosis of purkinje cells (arrow). (E) and (F) 1 LD50 3 day of abrin administered mice brain histology showed more severe changes in pyknotic neurons and necrosis respectively in granular layer as compared to 0.4 LD50.

being reported in human poisoning cases. Convulsions and episodes of seizures are frequently associated with consumption of Abrus seeds. Altered sensorium with depression of central nervous system also appears to be characteristic features. Reports in the past also have documented cardiac arrhythmias in severe poisoning (Pillay et al., 2005; Sahoo et al., 2008). Death of a patient with symptoms of acute demyelinating encephalopathy due to ingestion of abrin seeds was reported (Sahni et al., 2007). Since involvement of CNS is evident in case reports, we investigated neurodegenerative changes in mice after abrin exposure as very little information is available. The brain is especially vulnerable to ROS damage because of its high oxygen consumption rate, abundant lipid content, and relative paucity of antioxidant enzymes compared with other organs (Coyle and Puttfarcken, 1993). After internalization of abrin, along with protein synthesis inhibition, toxin also induces loss of

mitochondrial membrane potential which in turn leads to imbalance in ROS generation and neutralization, resulting in ROS overproduction. As oxidative stress is multi-signaling cascade, ROS overproduction due to abrin exposure leads to activation of several signaling pathways and leads to abrin toxicity (Narayanan et al., 2004). Oxidative stress mediated brain damage is noticed in mice treated with 0.4 and 1.0 LD50 dose characterized by increased levels of ROS in blood and brain, depletion of GSH and increased levels of lipid peroxidation in brain. Our earlier results in cultured cells exposed to ricin or abrin showed similar ROS production and GSH depletion which are initial events of oxidative cell damage (Bhaskar et al., 2008; Rao et al., 2005). Potential beneficial role of various antioxidant compounds in neurologic diseases was reported in studies involving in vitro and animal models (Delanty and Dichter, 2000). Antioxidant properties of gossypin contribute to neuroprotective actions in cultured

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Fig. 8. Photomicrographs of control, and abrin treated (0.4 and 1 LD50 dose) mice brain hippocampus H & E 40. (A) and (D) Control mice showing normal cellular composition arranged in three layers. (B) Section of mice brain with Abrin (0.4 LD50, 1 day) showing necrotic changes (arrow) in pyramidal cells of hippocampus. (C) Mice treated with Abrin (0.4 LD50, 3 day) showing more severe changes as compared to day 1. (E) and (F) 1 LD50 1 day and 3 day of abrin administered mice brain histology showed more severe necrotic changes in hippocampus region as compared to 0.4 LD50.

cortical cells by inhibiting oxidative stress mediated toxicity (Yoon et al., 2004). In our in vitro study in HeLa cells (Bhaskar et al., 2008), we proved reversal of oxidative stress with antioxidant NAC after abrin exposure. However, there are reports of irreversible neuronal damage in primary progressive and secondary progressive course of diseases like Multiple Sclerosis, as a consequence of oxidative and nitosative stress as well (Kornek et al., 2000; Wu and Tsirka, 2009). Till date there are no reports on the reversal of neurotoxic effects by antioxidants in studies involving abrin poisoning. ROS generation due to an unbalanced overproduction give rise to oxidative stress which can induce neuronal damage, ultimately leading to neuronal death by apoptosis or necrosis (Di Matteo and Esposito, 2003). AChE is a serine protease that hydrolyses the neurotransmitter acetylcholine. Principal form of AChE is found in neurons, neuromuscular junctions and erythrocyte membrane. AChE has some non-enzymatic and non-hydrolysing functions as well. It participates in the process of apoptosis. AChE plays a critical role in

the formation of apoptosome, a large quaternary protein structure formed in the process of apoptosis (Park et al., 2004). Serum cholinesterase is a circulating plasma glycoprotein synthesized in the liver including group of enzymes present in cerebrospinal fluid, liver, glial cells and plasma. Our results show significant inhibition of AChE in both brain and serum indicating involvement of both forms of AChE. Histopathology of brain confirms the damage to glial cells. There are no reports available for abrin mediated changes in neurotransmitters release and inhibition. Biogenic amines like 5HT, DA and NE in brain are important neurotransmitters controlling key functions of CNS. A marked alteration in their levels is observed during abrin toxicity relative to control. Many evidences show involvement of 5-HT in various depression pathologies, as MAO and 5-HT uptake inhibitors are widely used in antidepression therapy (Ban, 2001). The results obtained visualized increase in 5-HT and NE concentration and decrease

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in MAO activity at both the doses and time points, signifying central nervous system depression in mice exposed to abrin exposure. Alterations in metabolism of indoleamines lead to increase concentration of 5-HT, which results in decreased activity of MAO enzyme in brain. Abrin also may have direct inhibitory effect on MAO enzyme once it entered the cell. We may hypothesize that abrin toxin inhibits the synthesis of MAO enzyme leading to increase in 5-HT and NE concentration by interfering in indoleamines metabolism. Changes of any parameter of neurotransmission can be the result of neuronal death due to cytotoxic effects of the neurotoxicants. Abrin mediated brain microarray studies confirms the neurotoxic potential of abrin in mice (Bhaskar et al., 2012). The present results showed alterations in parameters of neurotoxicity. Exact mechanism involved in abrin mediated neurotoxicity is difficult to decipher as signaling pathways are not yet explored. We reported both dose and time-dependent transcriptional responses induced by abrin in the adult mouse after intraperintoneal exposure (Bhaskar et al., 2012). The microarray results determined a number of differentially expressed genes responsible for various activities, such as immune response, cell adhesion, chemotaxis, inflammatory processes, transcription and signal transduction. Enzymatically derived nitric oxide (NO) has been implicated in numerous physiological and pathological processes in the brain. The expression and activity of the inducible isoform of NO synthase (iNOS) play a pivotal role in sustained and elevated NO release. iNOS is a crucial enzyme that participates in chronic inflammation of the CNS. iNOS is usually expressed after inflammatory, neurodegenerative or ischemic brain damage in CNS (Heneka and Feinstein, 2001). Dose and time dependent increase in iNOS protein levels directly indicates neuroinflammation and neurodegeneration, also confirmed by histopathology results. Interaction of myelin sheath with cytosolic surfaces is maintained by myelin basic protein (Liu et al., 2012). We observed a dose dependent decrease in expression of MBP in brain. Demyelination is due to immune mechanisms underlying inflammatory neuropathy (Liu et al., 2012). A few reports exist that investigate mechanism of abrin induced apoptosis in vitro (Griffiths et al., 1987; Narayanan et al., 2004) and DNA damage (Bhaskar et al., 2008). But more studies are required to determine gene signature involved in the abrin induced intoxication process. The classic appearance of neuron degeneration is that seen in the process known as acute eosinophilic neuron degeneration. The degenerating neurons are characterized at the light microscopic level by cell body shrinkage, loss of nissl substance, intensely stained eosinophilic cytoplasm, and a small/shrunken darkly stained (pyknotic) nucleus that may eventually fragment (Garman, 2011). In an earlier study using 1 and 2 LD50 doses of abrin, we reported dose and time dependent neuroinflammatory damage indicated by infiltration of inflammatory cells into cortex region of brain. Abrin exposure resulted in the induction of rapid immune and inflammatory response in brain (Bhaskar et al., 2012). In this study as well histopathology results clearly showed similar appearance of degenerating neurons. This is in agreement with human fatal case of poisoning where MRI of the brain scan showed evidence of demyelinating encephalitis (Sahni et al., 2007). emyelination is immune-mediated and abrin is a well known immune modulator and stimulator (Sahni et al., 2007). In conclusion, overall results obtained in the current study signify abrin induced oxidative stress and neurotoxicity after abrin toxin exposure. Despite the deleterious effects of abrin on brain, very limited studies are reported. The present study provides new insights into the neurotoxic potential of abrin in terms of brain oxidative stress, lipid peroxidation, AChE inhibition, alteration in neurotransmitters, increased iNOS and decrease in MBP protein

expression. The sequale in abrin toxin mediated neutoxicity here is, oxidative stress leading to AChE inhibition, which in turn leads to alteration of neurotransmitters resulting in CNS depression and neurodegeneration by iNOS activation and depletion of MBP protein in brain. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document Transparency documents associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neuro. 2014.06.015. Acknowledgements We thank Prof (Dr) MP Kaushik, Director, Defence Research and Development Establishment for providing all facilities and Dr Pravin Kumar, Head, Pharmacology and Toxicology Division for constant support during this study. We thank Dr SC Pant for his help in histopathology analysis. Ms Bhavana is recipient of UGC Junior research fellowship. Ms Mona is recipient of Senior research fellowship from CSIR. This work was supported by the grant from Ministry of Defence, India References Ban TA. Pharmacotherapy of depression: a historical analysis. J Neural Transm 2001;108:707–16. Behl C. Amyloid beta-protein toxicity and oxidative stress in Alzheimer’s disease. Cell Tissue Res 1997;290:471–80. Bhaskar ASB, Deb U, Kumar O, Lakshmana Rao PV. Abrin induced oxidative stress mediated DNA damage in human leukemic cells and its reversal by N-acetylcysteine. Toxicol In Vitro 2008;22:1902–8. Bhaskar AS, Gupta N, Rao PV. Transcriptomic profile of host response in mouse brain after exposure to plant toxin abrin. Toxicology 2012;299(1):33–43. Boskovic M, Vovk T, Kores Plesnicar B, Grabnar I. Oxidative stress in schizophrenia. Curr Neuropharmacol 2011;9:301–12. Chiarugi P, Pani G, Giannoni E, Taddei L, Colavitti R, Raugei G, et al. Reactive oxygen species as essential mediators of cell adhesion: the oxidative inhibition of a FAK tyrosine phosphatase is required for cell adhesion. J Cell Biol 2003;161:933–44. Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders. Science 1993;262:689–95. Delanty N, Dichter MA. Antioxidant therapy in neurologic disease. Arch Neurol 2000;57:1265–70. Deshpande VR, Dubey PN, Rao MK. Toxicity of Abrus precatorius. Indian J Med Sci 1961;15:195–7. Dias V, Junn E, Mouradian MM. The role of oxidative stress in Parkinson’s disease. J Parkinson Dis 2013;3:461–91. Dickers KJ, Bradberry SM, Rice P, Griffiths GD, Vale JA. Abrin poisoning. Toxicol Rev 2003;22:137–42. Di Matteo V, Esposito E. Biochemical and therapeutic effects of antioxidants in the treatment of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Curr Drug Targets CNS Neurol Disord 2003;2:95–107. Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88–95. Franz H. The ricin story. In: Franz H, editor. Advances in lectin research, vol. 1. Berlin: Verlag Volk und Gesundheit; 1988. p. 10–25. Franz DR, Jaax NK. Ricin toxin. In: Medical aspects of chemical and biological warfare. Washington, DC: Borden Institute, Walter Reed Army Medical Center; 1997: 631– 42 (Chapter 32). Frohne D, Schmoldt A, Pfander HJ. Die Paternostererbse–Keineswegs harmlos. Dtsc Apothek Zeit 1984;124:2109–13. Garman RH. Histology of the central nervous system. Toxicol Pathol 2011;39:22–35. Griffiths GD, Leek MD, Gee DJ. The toxic plant proteins ricin and abrin induce apoptotic changes in mammalian lymphoid tissues and intestine. J Pathol 1987;151:221–9. Heneka MT, Feinstein DL. Expression and function of inducible nitric oxide synthase in neurons. J Neuroimmunol 2001;114:8–18. Hissin PJ, Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 1976;74:214–26. Howat AJ. The toxic plant proteins ricin and abrin induce apoptotic changes in mammalian lymphoid tissues and intestine. J Pathol 1988;154:2933. Hughes JN, Lindsay CD, Griffiths GD. Morphology of ricin and abrin exposed endothelial cells is consistent with apoptotic cell death. Hum Exp Toxicol 1996;15:443–51.

A.S.B. Bhasker et al. / NeuroToxicology 44 (2014) 194–203 Ischiropoulos H, Beckman JS. Oxidative stress and nitration in neurodegeneration: cause, effect, or association. J Clin Invest 2003;111:163–9. Jacobowitz DM, Richardson JS. Method for the rapid determination of norepinephrine, dopamine, and serotonin in the same brain region. Pharmacol Biochem Behav 1978;8:515–9. Kornek B, Storch MK, Weissert R, Wallstroem E, Stefferl A, Olsson T, et al. Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions. Am J Pathol 2000;157:267–76. Kumar O, Sugendran K, Vijayaraghavan R. Oxidative stress associated hepatic and renal toxicity induced by ricin in mice. Toxicon 2003;41:333–8. Kumar O, Kannoji A, Jayaraj R, Vijayaraghavan R. Purification and characterization of abrin toxin from white Abrus precatorius seeds. J Cell Tissue Res 2008;8:1243–8. Lord JM, Roberts LM, Robertus JD. Ricin: structure, mode of action, and some current applications. FASEB J 1994;8:201–8. Liu H, Shiryaev SA, Chernov AV, Kim Y, Shubayev I, Remacle AG, et al. Immunodominant fragments of myelin basic protein initiate T cell-dependent pain. J Neuroinflamm 2012;9:119. Narayanan S, Surolia A, Karande AA. Ribosome-inactivating protein and apoptosis: abrin causes cell death via mitochondrial pathway in Jurkat cells. Biochem J 2004;377:233–40. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. Olsnes S. The history of ricin, abrin and related toxins. Toxicon 2004;44:361–70. Ortiz GG, Pacheco-Moises FP, Bitzer-Quintero OK, Ramirez-Anguiano AC, Flores-Alvarado LJ, Ramirez-Ramirez V, et al. Immunology and oxidative stress in multiple sclerosis: clinical and basic approach. Clin Dev Immunol 2013;2013:708659.

203

Park SW, Vepachedu R, Owens RA, Vivanco JM. The N-glycosidase activity of the ribosome-inactivating protein ME1 targets single-stranded regions of nucleic acids independent of sequence or structural motifs. J Biol Chem 2004;279:34165–74. Patocˇka J, Strˇeda L. Plant toxic proteins and their current significance for warfare and medicine. J Appl Biomed 2003;1:141–7. Pillay VV, Bhagyanathan PV, Krishnaprasad R, Rajesh RR, Vishnupriya N. Poisoning due to white seed variety of Abrus precatorius. J Assoc Physicians India 2005;53:317–9. Rao PV, Jayaraj R, Bhaskar ASB, Kumar O, Bhattacharya R, Saxena P, et al. Mechanism of ricin-induced apoptosis in human cervical cancer cells. Biochem Pharmacol 2005;69:855–65. Sahni V, Agarwal SK, Singh NP, Sikdar S. Acute demyelinating encephalitis after jequirity pea ingestion (Abrus precatorius). Clin Toxicol (Phila) 2007;45:77–9. Sahoo R, Hamide A, Amalnath SD, Narayana BS. Acute demyelinating encephalitis due to Abrus precatorius poisoning – complete recovery after steroid therapy. Clin Toxicol (Phila) 2008;46:1071–3. Socci DJ, Bjugstad KB, Jones HC, Pattisapu JV, Arendash GW. Evidence that oxidative stress is associated with the pathophysiology of inherited hydrocephalus in the HTx rat model. Exp Neurol 1999 Jan;155(1):109–17. Stirpe F. Ribosome-inactivating proteins. Toxicon 2004;44:371–83. Wu M, Tsirka SE. Endothelial NOS-deficient mice reveal dual roles for nitric oxide during experimental autoimmune encephalomyelitis. Glia 2009;57:1204–15. Wurtman RJ, Axelrod J. A sensitive and specific assay for the estimation of monoamine oxidase. Biochem Pharmacol 1963 Dec;12:1439–41. Yoon I, Lee KH, Cho J. Gossypin protects primary cultured rat cortical cells from oxidative stress- and beta-amyloid-induced toxicity. Arch Pharm Res 2004;27:454–9.

Plant toxin abrin induced oxidative stress mediated neurodegenerative changes in mice.

Abrin is a potent plant toxin. It is a heterodimeric protein toxin which is obtained from the seeds of Abrus precatorius plant. At cellular level abri...
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