Lathyrus Sativus Neurotoxin Research Articles

In vitro activation of protein kinase C by beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, the Lathyrus sativus neurotoxin.

Beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (L-ODAP) toxicity has been associated with lathyrism; a spastic paraparesis caused by excessive dietary intake of the pulse Lathyrus sativus. We investigated the effect of Lathyrus neurotoxin L-ODAP on protein kinase C (PKC) activity under in vitro conditions. L-ODAP activated phosphorylation activity of purified chick brain PKC. Both lysine-rich (histone III-S) and arginine-rich (protamine sulfate) substrate phosphorylation was enhanced in the presence of L-ODAP. The activation is concentration dependent, and maximal activation is observed at 100 microM concentration. Protamine sulfate phosphorylation was enhanced by 47%, whereas histone III-S phosphorylation was enhanced by 50% over PS/PDBu/Ca2+ dependent activity. The nontoxic D-isomer (D-ODAP) did not affect both histone III-S and protamine sulfate phosphorylation activity. These results indicate that L-ODAP taken up by neuronal cells could also contribute to PKC activation and so be associated with toxicity.

Excitotoxicity of Lathyrus Sativus Neurotoxin in Leech Retzius Neurons

The effects of Lathyrus sativus neurotoxin were studied on the cell membrane potential and cellular cation composition in Retzius nerve cells of the leech Haemopis sanguisuga, with ion-selective microelectrodes using liquid ion-exchangers. Bath application of 10-4 mol/l Lathyrus sativus neurotoxin for 3 min depolarized the cell membrane potential and decreased the input resistance of directly polarized membrane in Retzius neurons. At the same time the cellular Na+ activity increased and cellular K+ activity decreased with slow but complete recovery, while the intracellular Ca2+ concentration was not changed. Na+-free Ringer solutions inhibited the depolarizing effect of the neurotoxin on the cell membrane potential. Zero-Ca2+ Ringer solution or Ni2+-Ringer solution had no influence on the depolarizing effect of the neurotoxin on the cell membrane potential. It is obvious that the increase in membrane conductance and depolarization of the cell membrane potential are due to an influx of Na+ into the cell accompanied by an efflux of K+ from the cell.

Differential effect of β- N-oxalylamino- l-alanine, the Lathyrus sativus neurotoxin, and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate on the excitatory amino acid and taurine levels in the brain of freely moving rats

We studied the effect of β-oxalylamino- l-alanine, a glutamate analog present in Lathyrus sativus seeds and implicated in the etiopathogenesis of neurolathyrism, and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate on the extracellular levels of aspartate, glutamate and taurine in the primary motor cortex of freely moving rats. We found that while both neurotoxins increase the level of aspartate and glutamate, only (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate is able to modulate the level of taurine. GYKI-52466, a non-competitive non-NMDA antagonist, inhibited β-oxalylamino- l-alanine-induced increase of aspartate, but not that of glutamate. Conversely, this antagonist proved to be very efficient in blocking the stimulating effect of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate on all three amino acids. We suggest that β-oxalylamino- l-alanine increases the level of glutamate in vivo by a mechanism not connected to its effect on the non-NMDA receptors, which might involve the inhibition of glutamate transport. This would allow the excitatory neurotransmitter to reach a concentration sufficient to stimulate the non-NMDA receptors, which in their turn mediate the specific release of aspartate. Although the role of aspartate as a neurotransmitter is still under discussion, it might indeed amplify the excitotoxic cascade through its action on NMDA receptors. We speculate that this sequence of events might represent an important step in the molecular cascade leading to the appearance of the selective motoneuron degeneration in neurolathyrism.

Receptor interactions of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, the Lathyrus sativus putative excitotoxin, with synaptic membranes.

Direct evidence for the excitotoxicity of 3-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin has been studied by examining the binding of chemically synthesized [2,3 3H]ODAP ([3H]ODAP) to synaptic membranes. [3H]ODAP binding to membranes was mostly nonspecific, with only a very low specific binding (15-20% of the total binding) and was also not saturable. The low specific binding of [3H]ODAP remained unaltered under a variety of assay conditions. A low Bmax of 3.2 +/- 0.4 pmol/mg and Kd 0.2 +/- 0.08 microM could be discerned for the high affinity interactions under conditions wherein more than 80-90% of the binding was nonspecific. While ODAP could inhibit the binding of [3H]glutamate to chick synaptic membranes with a Ki of 10 +/- 0.9 microM, even L-DAP, a non neurotoxic amino acid was also equally effective in inhibiting the binding of [3H]glutamate. The very low specific binding of [3H]ODAP to synaptic membranes thus does not warrant considering its interactions at glutamate receptors as a significant event. The results thus suggest that the reported in vitro excitotoxic potential of ODAP may not reflect its true mechanism of neurotoxicity.

Apoptosis induced by beta-N-oxalylamino-L-alanine on a motoneuron hybrid cell line.

It has been suggested that beta-N-oxalylamino-L-alanine, a non-protein amino acid present in the Lathyrus Sativus seeds, may play a role in the etiopathogenesis of neurolathyrism, a toxic form of motor neuron disease clinically characterized by a severe spastic paraparesis. In order to investigate the mechanisms of beta-N-oxalylamino-L-alanine-mediated cell death, we studied the effect of this neurotoxin as well as other excitatory amino acids agonists on the growth and survival of motoneuron hybrid ventral spinal cord 4.1 cells. beta-N-oxalylamino-L-alanine was toxic to ventral spinal cord 4.1 cells in a concentration-dependent fashion (0.5-10 mM). Among the excitatory amino acids tested, only glutamate (1-10 mM), quisqualate (1 mM) and, with less extent, beta-N-methylamino-L-alanine (10 mM) induced a significant reduction of cell survival. The effect of Lathyrus Sativus neurotoxin was a slow process, becoming apparent only after 24-48 h of incubation. Interestingly, a mathematical analysis applied to the time course and dose curve of beta-N-oxalylamino-L-alanine toxicity suggested that even for very low concentrations of the amino acid it is theoretically possible to predict a time-dependent effect. The cell death was not blocked by antagonists of N-methyl-D-aspartate or non-N-methyl-D-aspartate receptors; aurintricarboxylic acid and alpha-tocopherol gave a partial protection; cysteine (1 mM) prevented the toxic effect of both Lathyrus Sativus neurotoxin and glutamate as well as quisqualate. Morphologically, in the presence of either beta-N-oxalylamino-L-alanine, glutamate or quisqualate, ventral spinal cord 4.1 cells showed apoptotic features also confirmed by ISEL technique and agarose gel electrophoresis of genomic DNA. Thus, our results suggest that in ventral spinal cord 4.1 motoneuron hybrid cells, in the absence of functional synaptic excitatory amino acid receptors, beta-N-oxalylamino-L-alanine induces cell degeneration through an apoptotic mechanism, possibly mediated by a block of cystine/glutamate Xc antiporter.

Cloning and Characterization of a Gene Responsible for the Lathyrus sativus Neurotoxin Degradation

A plasmid pBYA1, capable of degrading the Lathyrus sativus neurotoxin β-N-oxalyl amino alanine (BOAA), was isolated from a soli microbe and a physical map of the plasmid constructed. A partial Sau3A library of the plasmid was then prepared in pUC18 and a recombinant clone with a 1.8 kb insert capable of growing in M9 medium, with BOAA as sole source of carbon and nitrogen, was isolated. A nested set of deletions of this 1.8 kb fragment was then generated using Bal31 and the shortened fragments subcloned in the vector pUC18. Each of these clones were sequenced by Sanger’s dideoxy method and the sequences overlapped and analysed. The 1.8 kb BOAA degrading fragment was found to contain a 630 nucleotide long coding stretch, coding for 191 amino acids. The initial 56 nucleotides were found to contain sequences resembling the Pribnow-Schaller box of prokaryotes, the “-35” sequences and a sequence resembling the “-43” region of the lac promoter.

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2.

Dispersed brain cells from 12-14 day old mouse embryos were loaded with the Ca2+-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca2+ concentration ([Ca2+]i) of 158 nM (SE +/- 5) in the presence of 1 mM [Ca2+]o. When external [Ca2+] was raised from 0 to 1 mM there was an increase of [Ca2+]i of 70 nM; with further additions of Ca to greater than or equal to 10 mM [Ca2+]o the level of [Ca2+]i increased by less than or equal to 25 nM. Releasable intracellular Ca2+ stores, estimated from the increase in [Ca2+] produced by 4-Br A23187 in the absence of extracellular Ca2+, were 24 fmol/10(6) cells. A small increase in [Ca2+]i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K+ was raised by 10-20 mM, intracellular Ca2+ levels increased from 152 (SE +/- 7) to 204 nM (SE +/- 10). These K+-induced increases in [Ca2+]i were blocked by verapamil, did not occur in the absence of extracellular Ca2+, and presumably reflect the activation of voltage-dependent Ca2+ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca2+]i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2-aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca2+-dependent and -independent mechanisms of action of these excitatory amino acids.

The Lathyrus sativus neurotoxin: Resistance of the squirrel monkey to prolonged oral high doses

Prolonged oral administration of the Lathyrus sativus neurotoxin, l-3-oxalylamino-2-aminopropionic acid (OAP), to three young male squirrel monkeys at dose rates of 0.6 to 6.0 mg/g body weight/day produced no neurologic signs and no adverse effects other than depressed activity and occasional foaming at the mouth. When the dose was increased to 8.0 to 8.5 mg/kg body weight/day in two animals, seizures and death occurred after 3 and 5 days. The signs were typical of acute OAP intoxication observed previously to occur in the squirrel monkey within an hour after a single ip dose (2 mg/g body weight) of OAP. Total cumulative dosage of OAP and duration of the experiments were as follows: Experiment 1, 9.5 g, 38 days; Experiment 2, 157 g, 178 days; Experiment 3, 64 g, 33 days. Histologic examination revealed no abnormalities in brain, spinal cord, or other organs. These experiments suggest that the adult squirrel monkey is highly resistant to chronic oral OAP intoxication. Under our experimental conditions, this species did not provide a satisfactory animal model for human neurolathyrism, a disease postulated to result from chronic OAP intoxication.

The Lathyrus sativus neurotoxin: Evidence of selective retention in monkey tissue

Epidemiological studies implicate the food toxin, l-3-oxalylamino-2-aminopropionic acid (OAP), in outbreaks of neurolathyrism. The disease occurs only in humans who have eaten excessive amounts of OAP-containing legumes for many months, thus suggesting that chronic exposure to OAP is needed to cause nerve damage and paralysis. These experiments were carried out to determine whether OAP might be selectively retained by certain tissues. With repeated exposure to OAP, tissues which selectively retain the toxin might build up sufficient concentrations to produce nerve damage. To investigate the rate of loss of OAP from animal tissue, we injected young squirrel monkeys with l-3-[ 14C]oxalylamino-2-aminopropionic acid and analyzed tissue for radioactivity 1, 24, and 72 hr later. Cerebellum was conspicuously different from other parts of brain in that the concentration of radioactivity (dpm/g) relative to brain as a whole increased with time. At 1 hr, the concentration in cerebellum was about the same as in brain as a whole; at 72 hr, it was more than twice as great. The half-life for disappearance of radioactivity from cerebellum (32.7 hr) matched that for bone and was the longest observed in these experiments. Unchanged [ 14C]OAP was the major component of radioactivity recovered from brain and other tissues. Entrapped blood in brain was negligible. These experiments suggest that cerebellum may selectively retain OAP and thus tend to accumulate neurotoxic concentrations. Cerebellum contains glutamate receptors, and OAP is a known glutamate antagonist.

Acute neurotoxicity of the Lathyrus sativus neurotoxin, l-3-oxalylamino-2-aminopropionic acid, in the squirrel monkey

This paper provides the first evidence that the Lathyrus sativus neurotoxin, l-3-oxalylamino-2-aminopropionic Acid (OAP), is toxic when administered intraperitoneally to a primate with a mature bloodbrain barrier. Drowsiness, vomiting, muscle tremors, twitching, convulsions, and death occurred in young male squirrel monkeys ( Saimiri sciureus) following injection of 4.26 and 11.4 μmol (0.750 and 2.00 mg) OAP/g body wt. Electroencephalographic changes characteristic of each stage of intoxication were observed. Unchanged OAP was recovered from brain of intoxicated animals. These observations support a possible role for OAP in the etiology of human neurolathyrism, a paralytic disease prevalent among adults in Central India who have consumed large quantities of L. sativus seeds for several months.

A sensitive and specific colorimetric method for the determination of α,β-diaminopropionic acid and the Lathyrus sativus neurotoxin

A sensitive color reaction of o-phthalaldehyde (OPT) with α,β-diaminopropionic acid (DAP) is described. The reaction is used to determine specifically the DAP content in tissues of the rat injected with this amino acid. The method indicated the presence in the liver and kidney of a alkali labile metabolite of DAP. A procedure is also described to determine the Lathyrus sativus neurotoxin β- N-oxalyl- l-α,β-diaminopropionic acid (ODAP) in tissues of the rat injected with the neurotoxin and also its content in the seeds. The procedure is rapid and would be of use in studies with DAP and the L. sativus neurotoxin.

STUDIES ON THE TISSUE AND SUBCELLULAR DISTRIBUTION OF β‐N‐OXALYL‐l‐α, β‐DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN

Abstract— β‐N‐Oxalyl‐l‐α, β‐diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin can be detected in significant concentrations in the synaptosomal fractions isolated from young rat brain and adult monkey spinal cord, when these animals manifest neurological symptoms after ODAP administration. However, isolated synaptosomes fail to exhibit any transport system for ODAP uptake. ODAP administered in vivo appears to get localized in a population of synaptosomes which exhibit a high affinity uptake system for glutamate.

L-2-Oxalylamino-3-aminopropionic acid, an isomer of Lathyrus sativus neurotoxin

The first chemical synthesis of l-2-oxalylamino-3-aminopropionic acid, an isomer of the Lathyrus sativus neurotoxin, is described. Studies on its biological properties are reported. Experiments with l-3-[ 14C-oxalyl]amino-2- aminopropionic acid show that the amount of 2-oxalylamino isomer detectable in seed extracts can be accounted for by rearrangement which occurs during isolation.

Biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid, the lathyrus sativus neurotoxin

The biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid (ODAP) the Lathyrus sativus neurotoxin has been found to follow the scheme depicted below: ▪The first reaction is catalysed by oxalyl-CoA synthetase which has properties similar to that of the enzyme in peas. The second reaction is catalysed by another enzyme which is specific to L. sativus and is designated as oxalyl-CoA-α,β-diaminopropionic acid oxalyl transferase. The enzymes have been purified by about 60-fold and their properties studied. A partial resolution of the two enzyme activities has been achieved using CM-sephadex columns.