Glufosinate ammonium induces convulsion 
through N-methyl-D-aspartate receptors in mice 

Neuroscience Letters 304 (2001) 123-125

Nobuko Matsumura, Chizuko Takeuchi, Keiichi Hishikawa, Tomoko Fujii, Toshio Nakaki*

Department of Pharmacology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan Received 27 January 2001; received in revised form 15 March 2001; accepted 21 March 2001

Abstract

Glufosinate ammonium, a broad-spectrum herbicide, causes convulsion in rodents and humans. Because of the structural similarities between glufosinate and glutamate, the convulsion induced by glufosinate ammonium may be ascribed to glutamate receptor activation. Three N-methyl-D-asparate (NMDA) receptor antagonists, dizocilpine, LY235959, and Compound 40, and an -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist, NBQX, were coadministrated with glufosinate ammonium (80 mg/kg, intraperitoneally) in mice. Statistical analyses showed that the NMDA receptor antagonists markedly inhibited the convulsions, while the AMPA/kainate receptor antagonist had no effect on the convulsion. These results suggest that the convulsion caused by glufosinate ammonium is mediated through NMDA receptors. 

Keywords: Convulsion; Glufosinate Ammonium; N-methyl-o-asparate; LY235959; Compound 40; dizocilpine; NBQX

* Corresponding author. Tel. + 81-3-3964-3793; fax: +81-33964-0602.
E-mail address: nakaki@med.teikyo-u.ac.jp  (T. Nakaki).

Glufosinate ammonium, a broad-spectrum herbicide, inhibits a plant glutamine synthetase leading to a complete inhibition of ammonia metabolism. However, even at the lethal dose of glufosinate ammonium glutamate and ammonia levels increase only slightly in mammals [9].The incidence of poisoning in humans caused by the ingestion of the glufosinate ammonium-containing herbicides is gradually increasing in Japan. This poisoning is characterized by various neurological symptoms such as the disturbances of consciousness, convulsions and apnea, which appear after an asymptomatic interval of several hours [15].The administration of glufosinate ammonium to the rat induces behavioral changes such as aggressiveness, wet dog shakes and limbic seizures [7,9]. It is reported that glufosinate ammonium induces apoptosis in the neuroepitherium of mouse embryos in culture [14].The mechanisms of these neurotoxicities of glufosinate ammonium are not clear. Owing to the structural analogy between glufosinate ammonium and glutamate, it's effect on glutamate receptor systems has been explored. Thus, glufosinate binds with L-[³H]-glutamate labeling sites in rat brain plasma membranes [6]. Glufosinate ammonium stimulates the production of nitric oxide, which was suppressed by an inhibitor of nitric oxide synthetase and NMDA receptor antagonists [11]. In this paper, we present a pharmacological characterization for the excitatory action of glufosinate ammonium, showing the involvement of NMDA receptors in the glufosinate ammonium-induced convulsion.

Specific pathogen-free 4-week-old male ddY mice were purchased from Sankyo Laboratory (Tokyo, Japan). The mice were placed in 35 X 25 X 17 cm cages. The cages were kept in a temperature-controlled room under a 12 h light-dark schedule. Drugs were dissolved in saline or water adjusted to pH 7.5, and then systematically administrated in mice by intraperitoneal injection. The results were expressed as the means + SE. A statistical analysis was performed by ANOVA, followed by the t-test. Product-limit estimates were performed by the Kaplan-Meier method, followed by the generalized Wilcoxon test. Glufosinate ammonium was obtained from Riedel-de Haen (Seeize, Germany), Dizocilpine was from Research Biochemicals International (Natick, MA). LY235959 (3S,4aR,6S,8aR-6-phosphonomethyl-decahydroisoquinoline-3-carboxylic acid) and Compound 40 (cis-l-amino-3(2-phosphonoacetyl)cyclobutane-l-carboxylic acid) were from Tocris Cookson Inc (Ballwin, MO). NBQX disodium (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium) was from Sigma Aldrich (Japan).

The various concentrations of glufosinate ammonium were intraperitoneally (i.p.) administrated in mice (30-100 mg/kg, i.p., n = 10 in each group) and then the occurrence of convulsion with duration of more than 2 s was observed for 350 min after the drug administration. The intraperitoneal administration of glufosinate ammonium at doses higher than 40 mg/kg caused convulsion in mice (Fig. 1). There was no difference in the onset of convulsion between 80 and 100 mg/kg. These observations are qualitatively consistent with a report that intracerebroventricular application of glufosinate ammonium causes general convulsion [9]. Furthermore we tested the ability of an NMDA receptor antagonist for preventing the convulsion. Dizocilpine was coadministrated (0.3 mg/kg, i.p.) with glufosinate ammonium (n = 10-1 1 in each group) in mice. This intraperitoneal dose of dizocilpine has been used for antagonizing the NMDA receptors [ 10]. Dizocilpine significantly prolonged the onset of convulsion caused by glufosinate ammonium at the dose of 80 and 100 mg/kg (Fig. 1).

Fig. 1. Dose-dependency of glufosinate ammonium for the onset of convulsions. 

The reciprocals of the onset time of convulsion were plotted on the ordinate. The concentrations of glufosinate ammonium were plotted on the abscissa. Filled circles , glufosinate ammonium alone (i.p.); open circles , glufosinate ammonium (i.p.) in combination with dizocilpine (0.3 mg/kg, i.p.) (n= 10-11 in each group). Abbreviations are: G, glufosinate ammonium; Diz, dizocilpine. Each point and bar represents the mean and SE. *P < 0.05 vs. the group treated with glufosinate ammonium and saline.

In order to elaborate on determining the subtype of glutamate receptors involved in the convulsion induced by glufosinate ammonium, four glutamate receptor antagonists were tested; dizocilpine [13] (0.3 mg/kg, i.p.), LY235959 [2,3] (5 mg/kg, i.p.), and Compound 40 [8] (5 mg/kg, i.p.), as NMDA receptor antagonists and NBQX [5,12] (30 mg/kg, i.p.), as an AMPA/kainate receptor antagonist. These doses were enough to antagonize the NMDA receptor or the AMPA/kainate receptor. The onset times and rate of the convulsion were plotted according to the Kaplan-Meier method. The inhibitory effects of the antagonists were shown as the delay of the onset time of convulsion (Fig. 2). The mean convulsion times were 157, 136, 252, 270, and 342 min, for saline, NBQX, dizocilpine, Compound 40, and LY235959, respectively. The marked delays of the onset of convulsion were observed in the NMDA receptor antagonists. The differences in the Kaplan-Meier curves between the saline and each antagonist were estimated by the generalized Wilcoxon test. The significant level of 0.05 was considered acceptable for statistical significance. The effects of the NMDA receptor antagonists (dizocilpine, LY235959, and Compound 40) were statistically significant (X^{2 =} 10.918, P < 0.001,  X² = 10.387, P = 0.001, and X² = 4.238, P = 0.040, respectively), compared with that of saline, while NBQX was not significant (X² = 1.949, P = 0.163). No difference was seen between saline and NBQX. Fig. 2 demonstrates clearly that the NMDA receptor antagonists are effective in reducing the convulsion, whereas the AMPA/kainate receptor antagonist was ineffective. These results suggest that NMDA receptor activation plays a major role in the onset of convulsion that is induced by glufosinate ammonium. This finding is consistent with the view that the stimulation of NMDA receptors causes the convulsion in mice [1,4].

Fig. 2. Kaplan-Meier analysis for the glufosinate ammonium-induced convulsion. 

The plot demonstrates the onset time and rate of convulsion after the drug injection. The convulsions were induced by glufosinate ammonium (80 mg/kg, i.p.) in combination with saline (n = 23), NBQX (30 mg/kg, i.p., n = 8), dizocilpine (Diz) (0.3 mg/kg, i.p., n = 11), Compound 40 (5 mg/kg, i.p., n = 6), and LY235959 (5 mg/kg, i.p., n = 6). The drugs were administrated at time zero. Only Diz, Compound 40 (P < 0.05) and LY235959 were statistically significant compared with saline.

We previously reported that glufosinate ammonium stimulates nitric oxide production through NMDA receptors [ 11 ]. There is a conspicuous difference in the mode of action of glufosinate ammonium between the nitric oxide production and the convulsion. Nitric oxide is produced in 10 min after the administration of glufosinate ammonium infused with the microdialysis probe [11]. In contrast, the intracerebroventricular application of glufosinate ammonium induced general convulsion approximately 3 h later [9]. A similar delay was observed by intraperitoneal administration in our study. The large time lag of the onset of convulsion appears to be a characteristic of glufosinate ammonium. A causal relationship between the two events remains elusive, although both events involve NMDA receptors. In conclusion, we suggest that NMDA receptors were involved in glufosinate ammonium-induced convulsion in mice. Although species difference has to be considered, this finding may contribute hopefully to the treatment of the glufosinate ammonium-induced convulsion of human cases.

This work was supported in part by Grant-in-Aid for Scientific Research (C) from the Japanese Ministry of Education, Science, Sports and Culture.

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