www.ToxicolRes.org Original Article Toxicological Research Toxicol. Res. Vol. 34, No. 3, pp. 267-279 (2018) https://doi.org/10.5487/TR.2018.34.3.267 Open Access Proteomic Changes in Chick Brain Proteome Post Treatment with Lathyrus Sativus Neurotoxin, β-N-Oxalyl-L-α,β-Diaminopropionic Acid (L-ODAP): A Better Insight to Transient Neurolathyrism Anil Kumar D1, Sumathi Natarajan1, Nabil A M Bin Omar1, Preeti Singh2, Rohan Bhimani3, and Surya Satyanarayana Singh1 1Department of Biochemistry, University College of Science, Osmania University, Hyderabad, Telangana, India 2Bharati Vidyapeeth Medical College, Pune, Maharashtra, India 3Hinduja Healthcare, Khar West, Mumbai, Maharashtra, India Abstract Neurolathyrism is a neurodegenerative disorder characterized by spastic paraplegia resulting from the excessive consumption of Lathyrus sativus (Grass pea). β-N-Oxalyl-L-α,β-diaminopropionic acid (L-ODAP) is the primary neurotoxic component in this pea. The present study attempted to evaluate the proteome-wide alterations in chick brain 2 hr and 4 hr post L-ODAP treatment. Proteomic analysis of chick brain homogenates revealed several pro- teins involved in cytoskeletal structure, signaling, cellular metabolism, free radical scavenging, oxidative stress and neurodegenerative disorders were initially up-regulated at 2 hr and later recovered to normal levels by 4 hr. Since L-ODAP mediated neurotoxicity is mainly by excitotoxicity and oxidative stress related dysfunctions, this study further evaluated the role of L-ODAP in apoptosis in vitro using human neuroblastoma cell line, IMR-32. The in vitro studies carried out at 200 μM L-ODAP for 4 hr indicate minimal intracellular ROS generation and alteration of mitochondrial membrane potential though not leading to apoptotic cell death. L-ODAP at low con- centrations can be explored as a stimulator of various reactive oxygen species (ROS) mediated cell signaling path- ways not detrimental to cells. Insights from our study may provide a platform to explore the beneficial side of L- ODAP at lower concentrations. This study is of significance especially in view of the Government of India lifting the ban on cultivation of low toxin Lathyrus varieties and consumption of this lentil. Key words: L-ODAP, Neurolathyrism, Chick brain proteome, Mitochondria membrane potential, Caspase, Apoptosis INTRODUCTION of grass pea triggers a characteristic form of spastic para- paresis known as neurolathyrism in animals and humans Grass pea (Lathyrus sativus L., Leguminosae) is a (5-7), its cultivation was banned in certain countries drought crop that is cultivated in parts of Asia, Middle (2,6,8). The agent responsible for this cortical motor neu- East, and Africa as food for humans and fodder for ani- ron disease appears to be β-N-oxalyl-L-α,β-diaminopropi- mals, owing to its nutritive value and economic signifi- onic acid (L-ODAP), also known as β-N-oxalyl-amino-L- cance (1-4). Since the prolonged and exclusive consumption alanine present in the seed as the free amino acid (9-14). Correspondence to: Anil Kumar D, Department of Biochemistry, This is an Open-Access article distributed under the terms of the University college of science, Osmania university, Hyderabad, Tel- Creative Commons Attribution Non-Commercial License (http:// angana 500007, India creativecommons.org/licenses/by-nc/3.0) which permits unre- E-mail: [email protected] stricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: ODAP, N-oxalyl-L-α,β-diaminopropionic acid; PEBP1, phosphatidylethanolamine binding protein 1; HIF, hypoxia inducible factor; ROS, reactive oxygen species; IEF, isoelectric focusing; ACN, acetonitrile; DCFH-DA, 2',7'-dichlorodihydrofluo- rescein diacetate; DCF, 2',7'-dichlorofluorescein. 267 plSSN: 1976-8257 eISSN: 2234-2753 268 Anil Kumar D et al. L. sativus, a drought-resistant crop, once widely grown metabolism, oxidative stress response, and those known to in Africa and Asia, was banned due to the outbreak of play roles in neurodegenerative disorders such as Alzhei- lathyrism. Ever since the discovery of L-ODAP as the mer’s and Parkinson’s diseases were initially up-regulated neurotoxin responsible for lathyrism, extensive studies at 2 hr and later returned to normal levels by 4 hr corrobo- have been conducted focusing on its mechanism of action, rating with features of transient lathyrism observed in reasons for its neurotoxicity, and neutralization of its toxic chicks. Further in vitro assays carried out with 200 μM L- effects by antioxidants (15). Previous studies report exci- ODAP on the human neuroblastoma cell line IMR-32 totoxicity and oxidative stress-related dysfunctions as par- indicated a 1.2-fold increase in intracellular reactive oxy- tial causes of ODAP-mediated neurotoxicity (16). Animal gen species (ROS) production and a 1.8-fold increase in studies have shown the α-isomer of ODAP (D-ODAP) to be cells with a depolarized mitochondrial membrane; how- less neurotoxic than the β-isomer (L-ODAP) (17) or non- ever, it did not affect apoptosis or the levels of active neurotoxic when introduced into the cerebrospinal fluid caspases. These findings open up avenues for evaluating (18). D-ODAP lacks neurotoxic properties when tested in L-ODAP as a stimulator of various ROS-mediated signal- tissue culture (14). The prolonged consumption of L- ing pathways without being toxic to cells, which can be ODAP and transport across the blood brain barrier indi- further explored. cates that it potentially affects the central nervous system and over time, results in motor neuron disease (19). MATERIALS AND METHODS Molecular mechanisms proposed to explain the neuro- toxicity of L-ODAP generally emphasize that its pharma- Materials. D-ODAP and L-ODAP were a generous cological and structural analogs act on the excitatory gift from Lathyrus technologies (Hyderabad, India). amino acid glutamate (20-22). L-ODAP exerts its effects Immobilized pH-gradient strips (pH range 3~10), car- by causing an imbalance in calcium homeostasis (23) and rier ampholyte (BiolyteTM 3~10) were purchased from free radical-mediated mitochondrial dysfunction (24). L- Bio-Rad Laboratories (CA, USA), a broad-range molecu- ODAP plays a prominent role in signaling, wherein it is lar marker was purchased from Genei (Bangalore, India), known to activate calcium-dependent protein kinase C (25), and all reagents required for 2D electrophoresis were pro- mimic conditions of hypoxia leading to the down-regulation cured from Sigma Aldrich (MO, USA). of phosphatidylethanolamine binding protein 1 (PEBP1), and activate hypoxia inducible factor (HIF)-1α (26). Experimental animals. Four-day-old white leghorn Incidences of neurolathyrism have declined remarkably male chicks (Gallus gallus domesticus) were purchased in the past few decades due to changes in economic condi- from Venkateswara Hatcheries Pvt. Ltd. (Hyderabad, India). tions and as part of balanced diet. Today, research on L- The experiments were conducted following approval from ODAP is mainly focused on the detoxification pathways the University Ethical Committee (383/01/a/CPCSEA) and positive aspects of homoarginine in grass pea (27); and comply with the laws of India. As our study required recent studies have focused on angiogenesis (28), vasodi- isolation of the brain for profiling protein expression, the lation, and adaptations to hypoxia (25,29,30). Patents filed chicks were killed by cervical dislocation and all efforts in recent years highlight the therapeutic value of this were made to minimize suffering. amino acid in hemorrhage, chronic hypoxia-induced neu- rodegeneration, thrombocytopenia, and oral cavity prob- Cell culture. Human neuroblastoma cell line IMR-32 lems. With the USA and China acquiring patents on ODAP was procured from National Centre for Cell Science, based on its therapeutic applications (29), development of Pune, India. The cells were cultured in minimum essential grass pea varieties with low ODAP content (31), and the medium (Sigma Aldrich) supplemented with 10% FBS recent lifting of the ban by the Indian government on the (PAN Biotech, Aidenbach, Germany), 2 mM L-glutamine, cultivation of grass pea (32), there is a new revolution in penicillin (100 U/mL), streptomycin (0.1 mg/mL), ampho- the way grass pea and lathyrism are perceived. Nutrient- tericin B (2.5 μg/mL), and nystatin (100 U/mL). Cells were rich food crops with minimal water demands such as grass grown and maintained in surface-treated cell culture ware o pea can play a critical role in alleviating global malnutrition. at 37 C in a humidified atmosphere with 5% CO2-95% air. Although current research is focused on tapping the Cells were made quiescent by overnight incubation in therapeutic potential of L-ODAP, there has not been any serum-free medium prior to stimulation with ODAP. study evaluating the effects of this molecule on brain pro- tein profiles. The present study aims to the compare the Treatment of four-day-old white leghorn male chicks brain proteome in four-day-old young white leghorn male with D- and L-ODAP. D-/L-ODAP was injected intra- chicks following the administration of ODAP with focus peritoneally (0.5 mg/g body weight) to white leghorn male on transient lathyrism and/or recovery. Proteins involved chicks (n = 6 chicks per group). The chicks were sacri- in cytoskeletal structure formation, signaling, cellular ficed by cervical dislocation at 2 and 4 hr, and the brains L-ODAP and Transient Neurolathyrism 269 were dissected. Control group consisted of untreated chicks. In-gel digestion of spots with trypsin. Proteins dif- ferentially regulated were excised, washed, and in-gel 2D electrophoresis. Control and ODAP-treated brains digested with trypsin. Briefly, protein spots of interest were washed in PBS followed by homogenization in a were picked from gels and transferred into microcentri- lysis buffer [100 mM Tris (pH 7.4), 200 mM NaCl, 0.1% fuge tubes. The spots were washed twice with 18.2 Mohm (v/v) Triton X-100, 2.5 mM EDTA, 2 mM ethylene gly- water for approximately 15 min.