Although organophosphorus agents are used worldwide as pesticides, there have been many reports of pesticide poisoning. Nerve agents are organophosphorus agents that interfere with neurotransmission and have been used as chemical weapons in wars. These agents mainly irreversibly inhibit the action of acetylcholinesterase, an enzyme that breaks down acetylcholine, a neurotransmitter, and are believed to cause acute symptoms of poisoning. However, in recent years, the presence of subacute, delayed toxicity independent of acetylcholinesterase inhibition has been reported for some organophosphorus agents. We analyzed the subacute and delayed toxicity of bis(isopropylmethyl)phosphonate (BIMP), which has the same phosphonate group as sarin. BIMP rounded out the morphology of the cells and reduced the proportion of cells in the G1 phase of the cell cycle over time. No DNA damage was observed, suggesting that BIMP may affect cell division.
BACKGROUND: Endothelial lipase (EL), a regulator of plasma high-density lipoprotein cholesterol (HDL-C), is secreted as a 68-kDa mature glycoprotein, and then cleaved by proprotein convertases. However, the clinical significance of the circulating EL fragments remains unclear. OBJECTIVE: The objective of this study was to analyze the impact of serum EL fragments on HDL-C levels and major adverse cardiovascular events (MACE). METHODS: Using novel monoclonal antibodies (RC3A6) against carboxy-terminal EL protein, we have established a new enzyme-linked immunosorbent assay (ELISA) system, which can detect both full-length EL protein (full EL) and carboxy-terminal truncated fragments (total EL) in serum. The previous sandwich ELISA detected only full EL. The full and total EL mass were measured in 556 patients with coronary artery disease. Among them, 272 patients who underwent coronary intervention were monitored for 2 years for MACE. RESULTS: There was a significant correlation between serum full and total EL mass (R = 0.45, P < .0001). However, the total EL mass showed a stronger inverse correlation with serum HDL-cholesterol concentration than the full EL mass (R = -0.17 vs -0.02). Kaplan-Meier analysis documented an association of serum total EL mass and MACE (log-rank P = .037). When an optimal cutoff value was set at 96.23 ng/mL, total EL mass was an independent prognostic factor for MACE in the Cox proportional hazard model (HR; 1.75, 95% CI; 1.10-2.79, P = .018). CONCLUSION: Serum total EL mass could be a predictor for MACE in patients with coronary artery disease. This novel ELISA will be useful for further clarifying the impact of EL on HDL metabolism and atherosclerosis.
Title: The Sarin-like Organophosphorus Agent bis (isopropyl methyl)phosphonate Induces Apoptotic Cell Death and COX-2 Expression in SK-N-SH Cells Arima Y, Yoshimoto K, Namera A, Makita R, Murata K, Nagao M Ref: Hiroshima J Med Sci, 65:1, 2016 : PubMed
Organophosphorus compounds, such as sarin, are highly toxic nerve agents that inhibit acetylcholinesterase (AChE), but not cholinesterase, via multiple mechanisms. Recent studies have shown that organophosphorus compounds increase cyclooxygenase-2 (COX-2) expression and induce neurotoxicity. In this study, we examined the toxicity of the sarin-like organophosphorus agent bis(isopropyl methyl)phosphonate (BIMP) and the effects of BIMP on COX-2 expression in SK-N-SH human neuroblastoma cells. Exposure to BIMP changed cell morphology and induced caspase-dependent apoptotic cell death accompanied by cleavage of caspase 3, caspase 9, and poly (ADP-ribose) polymerase (PARP). It also increased COX-2 expression, while pretreatment with a COX inhibitor, ibuprofen, decreased BIMP-dependent cell death and COX-2 expression in SK-N-SH cells. Thus, our findings suggest that BIMP induces apoptotic cell death and upregulates COX-2 expression.
Organophosphorus (OP) compounds such as sarin are toxic agents that irreversibly inhibit the enzyme acetylcholinesterase. A recent study showed that OP compounds also have multiple toxicity mechanisms, and another suggested that endoplasmic reticulum (ER) dysfunction contributes to OP toxicity. However, the signaling pathway and mechanisms involved are poorly understood. We examined whether the sarin-like OP agent bis(isopropyl methyl)phosphonate (BIMP), which exhibits toxicity similar to that of sarin, induced ER stress in human astrocytoma CCF-STTG1 cells. Our results demonstrate that BIMP exposure reduced cell viability. Moreover, it induced changes in mitochondrial membrane potential and increased cleavage of caspase 3. Treatment with BIMP increased the mRNA levels of the ER stress marker genes binding immunoglobulin protein (BiP) and the transcription factor C/EBP homologous protein (CHOP). Furthermore, BIMP increased the protein expressions and phosphorylation of BiP, CHOP, and protein kinase RNA-like ER kinase and the phosphorylation of eukaryotic translation initiation factor 2. Compared to BIMP treatment alone, pretreatment with the CHOP siRNA, siCHOP, decreased BIMP-dependent CHOP expression and improved CCF-STTG1 cell viability. Our findings suggest that BIMP induced mitochondrial dysfunction and apoptotic cell death event mediated by ER stress in CCF-STTG1 cells and that treatment targeted at managing ER stress has the potential to attenuate the toxicity of OP nerve agents.
The organophosphorus compound sarin irreversibly inhibits acetylcholinesterase. We examined the acute cardiovascular effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate (BIMP), in anaesthetized, artificially ventilated rats. Intravenous administration of BIMP (0.8mg/kg; the LD50 value) induced a long-lasting increase in blood pressure and tended to increase heart rate. In rats pretreated with the non-selective muscarinic-receptor antagonist atropine, BIMP significantly increased both heart rate and blood pressure. In atropine-treated rats, hexamethonium (antagonist of ganglionic nicotinic receptors) greatly attenuated the BIMP-induced increase in blood pressure without changing the BIMP-induced increase in heart rate. In rats treated with atropine plus hexamethonium, intravenous phentolamine (non-selective alpha-adrenergic receptor antagonist) plus propranolol (non-selective beta-adrenergic receptor antagonist) completely blocked the BIMP-induced increases in blood pressure and heart rate. In atropine-treated rats, the reversible acetylcholinesterase inhibitor neostigmine (1mg/kg) induced a transient increase in blood pressure, but had no effect on heart rate. These results suggest that in anaesthetized rats, BIMP induces powerful stimulation of sympathetic as well as parasympathetic nerves and thereby modulates heart rate and blood pressure. They may also indicate that an action independent of acetylcholinesterase inhibition contributes to the acute cardiovascular responses induced by BIMP.
On March 20, 1995, the Tokyo subway system was subjected to a horrifying terrorist attack with sarin gas (isopropyl methylphosphonofluoridate) that left 12 persons dead and over 5000 injured. In order to diagnose the definite cause of death of the victims, a new method was developed to detect sarin hydrolysis products in the erythrocytes and formalin-fixed cerebella from four victims of sarin poisoning. Sarin-bound acetylcholinesterase (AChE) was solubilized from the specimens of sarin victims and digested with trypsin. The sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion. The digested sarin hydrolysis products were subjected to trimethylsilyl derivatization and detected by gas chromatography-mass spectrometry. Sarin hydrolysis products were detected in all sarin poisoning victims.
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 new method was developed to detect sarin hydrolysis products from erythrocytes of four victims of sarin (isopropylmethylphosphonofluoridate) poisoning resulting from the terrorist attack on the Tokyo subway. Sarin-bound acetylcholinesterase (AChE) was solubilized from erythrocyte membranes of sarin victims, digested with trypsin, the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion, and the digested sarin hydrolysis products were subjected to trimethylsilyl derivatization and detected by gas chromatography-mass spectrometry. Isopropylmethylphosphonic acid, which is a sarin hydrolysis product, was detected in all sarin poisoning, victims we examined and methylphosphonic acid, which is a sarin and soman hydrolysis product, was determined in all victims. Postmortem examinations revealed no macroscopic and microscopic findings specific to sarin poisoning and sarin and its hydrolysis products were almost undetectable in their blood. We think that the procedure described below will be useful for the forensic diagnosis of acute sarin poisoning.
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.
Title: Molecular cloning of cDNA for the catalytic subunit of rat liver type 2A protein phosphatase, and detection of high levels of expression of the gene in normal and cancer cells Kitagawa Y, Tahira T, Ikeda I, Kikuchi K, Tsuiki S, Sugimura T, Nagao M Ref: Biochimica & Biophysica Acta, 951:123, 1988 : PubMed
A cloned cDNA encoding a catalytic subunit of type 2A protein phosphatase from a rat liver cDNA library was obtained by use of a synthetic oligonucleotide corresponding to the tryptic peptide sequence of the purified enzyme. There was only a single amino acid difference between the deduced amino acid sequence of the clone obtained and those of the catalytic subunits, 2A alpha, of the rabbit skeletal muscle, porcine kidney and human liver enzymes, suggesting that this clone was a rat 2A alpha cDNA. On Northern blot analysis using a cDNA fragment as a probe, three mRNA species were detected in rat liver: a major mRNA of 2.0 kb and a minor one of 2.7 kb under high stringency conditions, and also a 1.1 kb mRNA under low stringency conditions. The 2A alpha gene was found to be highly expressed in various tissues of rat, especially the brain. High levels of expression of the gene were also detected in mouse NIH3T3 cells and their transformants, and in human cancer cell lines as well as a human immortalized cell line.
