Increased levels of nitrogen oxides and lipid peroxidation in the rat brain after soman-induced seizures.

We have investigated the effect of soman-induced seizures on rat brain levels of nitrogen oxides (NOx) and lipid peroxidation (LPO) 30 min and 24 h after intoxication. Following administration of soman (90 microg/kg s.c.), acetylcholinesterase activity was reduced to <10% of control after 30 min, whereas some de novo synthesis had occurred after 24 h. Significant increases in the LPO products malondialdehyde (MDA) and (E)-4-hydroxy-2-nonenal (4-HNE) were seen in the cortex, hippocampus, striatum, thalamus and medulla-pons 30 min after administration. A significant increase in the brain NOx levels, suggesting an increase in NO production, was seen in the cortex after 30 min and in the hippocampus and the striatum after 24 h. No significant changes were observed in cerebellum. These data suggest the possibility that free radical reactions may be a primary cause of neuronal degeneration after soman intoxication.

Enzyme-based microassay for accurate determination of soman in blood samples.

Successful medical therapy for nerve agent intoxication requires early diagnosis and treatment. Current clinical diagnostic methods do not permit early or definitive confirmation of intoxication. To improve the chances of successful medical therapy against nerve agent intoxication, a sensitive enzyme-based microassay for rapid and accurate quantification of residual soman levels in blood was developed. The new analytical technique is based on the linear correlation between residual eel acetylcholinesterase activities and the inhibitor concentration. Blood samples were deproteinized with perchloric acid, followed by immediate neutralization after deproteinization. The mixtures were centrifuged at 3000g and the supernatant was directly assayed for soman. The sensitivity of the technique (18-1820 pg/ml blood) is comparable to that attained by GC-FID analysis (250 pg/ml blood). To facilitate routine analysis, the linear range of the assay was optimized to span over a factor of 100 (0.1-10 nM), with a typical correlation factor of at least 0.999 (six standards). The assay accuracy, checked with four different concentrations of soman, was within +/- 10%. The assay capability in monitoring the pharmacokinetic of soman was validated using both in vitro and in vivo rat models.

Correlation between cortical EEG and striatal microdialysis in soman-intoxicated rats.

In vivo microdialysis and EEG recording have been used in order to study the combined neurochemical and electrophysiological events during intoxication with soman (o-1,2,2-trimethylpropyl methylphosphono-fluoridate), a potent inhibitor of acetylcholinesterase (AChE), in the freely moving rat. All rats exposed to soman exhibited signs of AChE inhibition. The duration of EEG recorded seizures after soman intoxication averaged 43 +/- 24 min. The extracellular striatal levels of dopamine and GABA, increased significantly during the EEG seizure periods. Using an EEG based differentiation between seizure and non-seizure conditions, we found that intrastriatal release of dopamine, but not glutamate, during soman intoxication is highly correlated with seizures. Our results suggest that excitatory amino acids (EAA) involvement in soman-induced seizures, as demonstrated in hippocampus, may not be relevant in the striatum. Our data, instead, may indicate the importance of dopamine as a neurotoxic agent.

Release of dopamine, GABA and EAA in rats during intrastriatal perfusion with kainic acid, NMDA and soman: a comparative microdialysis study.

There is an increasing amount of experimental evidence that excitatory amino acids (EAAs) are involved in the brain lesions observed after severe intoxication with the highly toxic organophosphorus compound soman. This study was undertaken to compare the acute actions of soman, and the glutamatergic receptor agonists kainic acid and N-methyl-D-aspartate (NMDA) on striatal release of dopamine and amino acids. The neurotoxic compounds were administered in high (10 mM) concentrations by unilateral intrastriatal microdialysis perfusion in freely moving rats. During the microdialysis the animals were observed for toxic signs related to convulsion. The glial fibrillary acidic protein (GFAP) was monitored as a marker of neurotoxicity in parts of prefrontal cortex, hippocampus, striatum and cerebellum. Acetylcholinesterase (AChE) inhibition in six brain regions was measured after soman perfusion in order to assess its cerebral distribution. We found that soman perfusion induced a major release of dopamine, GABA and aspartate in the striatum. Kainic acid also induced a release of dopamine and aspartate. NMDA was not as potent an inducer of striatal neurotransmitter release as soman and kainic acid. Soman and kainic acid perfusion produced convulsive behaviour in the rats. The main neurochemical event in the striatum during soman- and kainate-induced convulsions is the release of dopamine. We suggest that this major dopamine release might be as important as an increase in EAA in the cascade of pathological events leading to the brain damage in the striatum observed after soman intoxication.

Measurement of the oxime HI-6 after peripheral administration in tandem with neurotransmitter levels in striatal dialysates: effects of soman intoxication.

In the present study, the technique of microdialysis combined with tandem high-performance liquid chromatography was used to determine the striatal levels of HI-6 and neurotransmitters following peripheral administration of HI-6 (50 mg/kg i.m.) in conscious, freely moving rats. The results were compared with those obtained in animals given soman (135 micrograms/kg i.p.) 1 min before HI-6 (50 mg/kg i.m.). Principal component analysis was used to study the effects of the different treatments on neurotransmitters and signs of poisoning. In all animals given HI-6, maximum levels of HI-6 appeared in the second 20-min fraction after administration of HI-6, then gradually declined, reaching the lower limits of detection after 3 hr. There was a correlation between severity of poisoning and neurochemical changes observed; dopamine and GABA levels increased as the severity of signs of poisoning increased. These results clearly demonstrate that HI-6 can penetrate into the brain of control and soman-intoxicated animals. Tandem measurement of dopamine electrochemically and HI-6 by UV detection provides a simple method for obtaining data on HI-6 penetration into the brain in neurochemical studies of soman poisoning and its treatment.

Estimation of the convulsive effect of cyanide in rats.

Convulsions are frequently observed in severe, acute cyanide intoxication. The mechanisms involved are not well known. In the present study, the convulsive effect of cyanide was examined in the rat by means of a dose threshold determination after infusion of cyanide. An optimal dose rate of 1.80 mg/min./kg was determined. By infusing this optimal dose rate it was possible to divide another cyanide-treated group of rats into two groups: one without and another with convulsions. After the experiment all animals were killed by exposure to high intensity microwave irradiation. Regional dopamine, noradrenaline and main dopamine metabolites were isolated and analyzed using high pressure liquid chromatography and electrochemical detection. Striatal noradrenaline was decreased but dopamine and its metabolites were increased in rats infused with cyanide at the optimal dose rate until the convulsions started. Animals infused with the threshold dose at the optimal dose rate did not show any significant changes in noradrenaline or in dopamine metabolites, but all animals showing convulsions also had increased striatal dopamine levels. Thus changes in dopamine appear to be dependent on convulsions.

The effects of cyanide on the extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-hydroxyindoleacetic acid and inositol phospholipid breakdown in the brain.

The effects of sodium cyanide on the extracellular levels of dopamine and the main brain metabolites of dopamine and 5-hydroxytryptamine were studied in awake, freely moving rats using microdialysis technique. Already 20 min after the administration of cyanide (2 mg/kg; ip) the extracellular level of striatal dopamine was increased and after 40 min an enhancement of the homovanillic acid level was observed. Slight but significant decreases of 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid were also observed. The increased extracellular levels of dopamine and homovanillic acid could indicate an increased release of dopamine. An alternative explanation could be an extracellular leakage of dopamine due to neuronal damage caused by cyanide. Cyanide has also been reported to induce marked changes in cytosolic brain Ca2+. We examined the effect of cyanide on inositol phospholipid breakdown in rat cerebral cortex and pig striatum in vitro. This breakdown was not affected by sodium cyanide and appears therefore not to be responsible for the reported changes of cytosolic Ca2+ induced by sodium cyanide.

Effects of cyanide in vitro on the activity of monoamine oxidase in striatal tissue from rat and pig.

We have shown previously in the rat that lethal, acute cyanide intoxication dramatically decreased the levels of dopamine (DA) in the striatum, while the synthesis of DA was increased. The main brain metabolite of DA, homovanillic acid, was also diminished. However, the levels of the oxidatively deaminated metabolite of DA, 3,4-dihydroxyphenylacetic acid, were not significantly changed. In order to elucidate further these findings we examined the effects in vitro of sodium cyanide on rat and pig brain monoamine oxidase (MAO; EC 1.4.3.4). The MAO activity was measured radiochemically using [14C]5-hydroxytryptamine (5-HT; 100 microM), [14C]phenethylamine (PEA; 20 microM) and [14C]DA (100 microM) as substrates. The amounts of cyanide added were comparable to those tissue concentrations of cyanide usually considered to be fatal in rats. The effect of cyanide on MAO was immediate. In rat, as well as pig, striatal tissue we found that cyanide produced a dose-dependent increase in the activity of MAO-A (as measured with 5-HT), but not MAO-B (as measured with PEA). The change in MAO activity was also seen with DA as substrate (MAO-A and -B). Kinetic constants, Km and Vmax, were determined. In both rat and pig striatum the Vmax values for 5-HT were significantly increased, but the values for PEA were not affected. A significant decrease in the Km value for PEA was, however, found in the presence of high concentrations of cyanide.

Effects of cyanide on the striatal dopamine receptor binding in the rat.

In previous studies we have shown that sodium cyanide decreases the striatal dopamine levels within 60 s compared with the controls. Treatment with sodium cyanide also increases the naturally occurring 3,4-dihydroxy-L-phenylalanine (L-DOPA) in the striatum, but not in any other region studied. An increase in the in vivo synthesis of dopamine in cyanide-treated rats has also been observed. In order to further elucidate the effects on the central dopaminergic pathways the effects of sodium cyanide on the striatal dopamine D1 and dopamine D2 receptor binding were studied in vitro and after administration of sodium cyanide. In the rats injected with sodium cyanide (2.0 mg/kg, i.p.) the Bmax of the striatal dopamine D1 receptor binding was significantly decreased 15 min and 1 h after the treatment. The striatal dopamine D2 receptor binding was decreased only at 1 h after the cyanide administration. Neither sodium cyanide nor its metabolite sodium thiocyanate did significantly change the striatal dopamine D1 and D2 receptor binding in vitro. Accordingly sodium cyanide and sodium thiocyanate do not have direct effects on the dopamine receptors studied. The effects of cyanide on dopamine D1 and D2 receptors are probably in part due to the effect of cyanide on the release of dopamine.