We report that there is a time-related change in the phospholipase C (PLC) activities of rat brain cytosol and membrane fractions after iv injection of a soman-like or a sarin-like organophosphorous agent (bis(isopropyl methyl)phosphonate [BIMP] and bis(pinacolyl methyl)phosphonate [BPMP]). PLCgamma was activated in the brain cytosol fraction from BPMP-injected rats. The phosphorylating activity of rat brain membrane fractions were enhanced by BPMP treatment. The brain membrane fractions from BPMP-treated rats phosphorylated several proteins, including supposedly PLCgamma in the brain cytosol fraction from control rats in vitro. These results suggest that soman and sarin may stimulate a membrane tyrosine kinase, including growth factor receptors, directly or indirectly.
One of the hydrolysis products of sarin (isopropyl methylphosphonofluoridate) was detected in formalin-fixed brain tissues of victims poisoned in the Tokyo subway terrorist attack. Part of this procedure, used for the detection of sarin hydrolysis products in erythrocytes of sarin victims, has been described previously. The test materials were four individual cerebellums, which had been stored in formalin fixative for about 2 years. Sarin-bound acetylcholinesterase (AChE) was solubilized from these cerebellums, purified by immunoaffinity chromatography, and digested with trypsin. Then the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion, subjected to trimethylsilyl derivatization (TMS), and detected by gas chromatography-mass spectrometry. Peaks at m/z 225 and m/z 240, which are indicative of TMS-methylphosphonic acid, were observed within the retention time range of authentic methylphosphonic acid. However, no isopropyl methylphosphonic acid was detected in the formalin-fixed cerebellums of these 4 sarin victims, probably because the isopropoxy group of isopropyl methylphosphonic acid underwent chemical hydrolysis during storage. This procedure will be useful for the forensic diagnosis of poisoning by protein-bound, highly toxic agents, such as sarin, which are easily hydrolysed. This appears to be the first time that intoxication by a nerve agent has been demonstrated by analyzing formalin-fixed brains obtained at autopsy.
A sarin-like organophosphorus agent, [bis(isopropyl methyl)phosphonate; BIMP], was synthesized. This agent has the same phosphonate group as sarin and also has the same anti-acetylcholinesterase activity potency as sarin. The ID50 and LD50 values of BIMP in mice after intravenous injection were 3.9 nM and 0.8 mg/kg, respectively. The AChE activities of their red blood cells and brains were dose-dependently reduced by intravenous BIMP. After preparation of experimental BIMP-exposed human red blood cells, BIMP-bound acetylcholinesterase (AChE) was solubilized from erythrocyte membranes, purified by immunoaffinity chromatography, digested with trypsin, and the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion. The digested sarin hydrolysis products were subjected to trimethylsilyl (TMS) derivatization and detected by gas chromatography-mass spectrometry. Isopropyl methylphosphonic- and methylphosphonic acids, which are the sarin hydrolysis products, were detected in experimental BIMP-exposed human red blood cells. This new method, which enables sarin's hydrolysis products to be detected in BIMP-exposed erythrocytes, is a useful tool for studying sarin-poisoning victims.
