Multifunctional compounds lithium chloride and methylene Blue attenuate the negative effects of diisopropylfluorophosphate on axonal transport in rat cortical neurons.

Organophosphates (OPs) are valuable as pesticides in agriculture and for controlling deadly vector-borne illnesses; however, they are highly toxic and associated with many deleterious health effects in humans including long-term neurological impairments. Antidotal treatment regimens are available to combat the symptoms of acute OP toxicity, which result from the irreversible inhibition of acetylcholinesterase (AChE). However, there are no established treatments for the long-term neurological consequences of OP exposure. In addition to AChE, OPs can negatively affect multiple protein targets as well as biological processes such as axonal transport. Given the fundamental nature of axonal transport to neuronal health, we rationalized that this process might serve as a general focus area for novel therapeutic strategies against OP toxicity. In the studies described here, we employed a multi-target, phenotypic screening, and drug repurposing strategy for the evaluations of potential novel OP-treatments using a primary neuronal culture model and time-lapse live imaging microscopy. Two multi-target compounds, lithium chloride (LiCl) and methylene blue (MB), which are FDA-approved for other indications, were evaluated for their ability to prevent the negative effects of the OP, diisopropylfluorophosphate (DFP) on axonal transport. The results indicated that both LiCl and MB prevented DFP-induced impairments in anterograde and retrograde axonal transport velocities in a concentration dependent manner. While in vivo studies will be required to confirm our in vitro findings, these experiments support the potential of LiCl and MB as repurposed drugs for the treatment of the long-term neurological deficits associated with OP exposure (currently an unmet medical need).

Corticosterone primes the neuroinflammatory response to DFP in mice: potential animal model of Gulf War Illness.

Gulf War Illness (GWI) is a multi-symptom disorder with features characteristic of persistent sickness behavior. Among conditions encountered in the Gulf War (GW) theater were physiological stressors (e.g., heat/cold/physical activity/sleep deprivation), prophylactic treatment with the reversible AChE inhibitor, pyridostigmine bromide (PB), the insect repellent, N,N-diethyl-meta-toluamide (DEET), and potentially the nerve agent, sarin. Prior exposure to the anti-inflammatory glucocorticoid, corticosterone (CORT), at levels associated with high physiological stress, can paradoxically prime the CNS to produce a robust proinflammatory response to neurotoxicants and systemic inflammation; such neuroinflammatory effects can be associated with sickness behavior. Here, we examined whether CORT primed the CNS to mount neuroinflammatory responses to GW exposures as a potential model of GWI. Male C57BL/6 mice were treated with chronic (14 days) PB/ DEET, subchronic (7-14 days) CORT, and acute exposure (day 15) to diisopropyl fluorophosphate (DFP), a sarin surrogate and irreversible AChE inhibitor. DFP alone caused marked brain-wide neuroinflammation assessed by qPCR of tumor necrosis factor-α, IL6, chemokine (C-C motif) ligand 2, IL-1ß, leukemia inhibitory factor, and oncostatin M. Pre-treatment with high physiological levels of CORT greatly augmented (up to 300-fold) the neuroinflammatory responses to DFP. Anti-inflammatory pre-treatment with minocycline suppressed many proinflammatory responses to CORT+DFP. Our findings are suggestive of a possible critical, yet unrecognized interaction between the stressor/environment of the GW theater and agent exposure(s) unique to this war. Such exposures may in fact prime the CNS to amplify future neuroinflammatory responses to pathogens, injury, or toxicity. Such occurrences could potentially result in the prolonged episodes of sickness behavior observed in GWI. Gulf War (GW) veterans were exposed to stressors, prophylactic medicines and, potentially, nerve agents in theater. Subsequent development of GW Illness, a persistent multi-symptom disorder with features characteristic of sickness behavior, may be caused by priming of the CNS resulting in exaggerated neuroinflammatory responses to pathogens/insults. Nerve agent, diisopropyl fluorophosphate (DFP), produced a neuroinflammatory response that was exacerbated by pre-treatment with levels of corticosterone simulating heightened stressor conditions. While prophylactic treatments reduced DFP-induced neuroinflammation, this effect was negated when those treatments were combined with corticosterone.

Discovery of non-oxime reactivators using an in silico pharmacophore model of oxime reactivators of OP-inhibited acetylcholinesterase.

We earlier reported an in silico pharmacophore model for reactivation of oximes to tabun-inhibited AChE. Since DFP (diisopropylfluorophosphate) like tabun is a G-agent simulator, we utilized the model as a rational strategy to discover non-oxime reactivators of DFP-inhibited AChE in this study. The phramacophore was used for virtual screening of two commercial databases, Maybridge and ChemNavigator, to identify reactivators which lack the oxime functions. The procedure led us to identify several potent non-oxime compounds that reactivate DFP-inhibited AChE. These non-oxime reactivators contain a nucleophile group in lieu of the oxime moiety in the compound. Five of these novel non-oximes showed Kr values within ten-fold of 2-PAM in an in vitro assay. The pharmacophore model contained a hydrogen bond acceptor, a hydrogen bond donor, and an aromatic ring features distributed in a 3D space. Calculated stereoelectronic properties reported earlier with respect to the location of molecular orbitals and electrostatic potentials were consistent with the model and the newly identified compounds. Down selection of compounds after virtual screening was performed on the basis of fit score to the model, conformational energy, and in silico evaluations for favorable blood-brain barrier (BBB) penetrability, octanol-water partition (log P), and toxicity (rat oral LD(50)) assessments. In vitro reactivation efficacy of the compounds was evaluated in a DFP-inhibited eel acetylcholinesterase assay.

Fullerene antioxidants decrease organophosphate-induced acetylcholinesterase inhibition in vitro.

Although organophosphate (OP)-induced acetylcholinesterase (AChE) inhibition is the critical mechanism causing toxicities that follow exposure, other biochemical events, including oxidative stress, have been reported to contribute to OP toxicity. Fullerenes are carbon spheres with antioxidant activity. Thus, we hypothesized that fullerenes could counteract the effects of OP compounds and tested this hypothesis using two in vitro test systems, hen brain and human neuroblastoma SH-SY5Y cells. Cells were incubated with eight different derivatized fullerene compounds before challenge with paraoxon (0=control, 5×10(-8), 10(-7), 2×10(-7) or 5×10(-7) M) or diisopropylphosphorofluoridate (DFP, 0=control, 5×10(-6), 10(-5), 2×10(-5), and 5×10(-5) M) and measurement of AChE activities. Activities of brain and SH-SY5Y AChE with OP compounds alone ranged from 55-83% lower than non-treated controls after paraoxon and from 60-92% lower than non-treated controls after DFP. Most incubations containing 1 and 10 µM fullerene derivatives brought AChE activity closer to untreated controls, with improvements in AChE activity often >20%. Using dissipation of superoxide anion radicals as an indicator (xanthine oxidation as a positive control), all fullerene derivatives demonstrated significant antioxidant capability in neuroblastoma cells at 1 µM concentrations. No fullerene derivative at 1 µM significantly affected neuroblastoma cell viability, when determined using either Alamar Blue dye retention or a luminescent assay for ATP production. These studies suggest that derivatized fullerene nanomaterials have potential capability to ameliorate OP-induced AChE inhibition resulting in toxicities.

Toxicity and characterization of cholinesterase-inhibition induced by diisopropyl fluorophosphate in Artemia salina larvae.

The acute toxicity of diisopropyl fluorophosphate (DFP) on three age classes of Artemia salina was evaluated. An increase in toxicity of this organophosphorous (OP) compound was found following longer development of A. salina larvae. The effects of pretreatment with the non-selective muscarinic antagonist atropine, the two reversible acetylcholinesterase inhibitors physostigmine and pyridostigmine, and the cholinesterase-reactivating oxime 2-pyridine aldoxime methoiodide (2-PAM), as individual and combined pretreatments, on DFP-induced lethality in 24h Artemia were also investigated. The lethal action of DFP was not prevented by pretreatment of 24h Artemia with atropine, physostigmine, and pyridostigmine, while 2-PAM proved effective against intoxication with this OP compound. The inhibitory effects of combinations of atropine (10(-5)M) plus 2-PAM or physostigmine were greater than those elicited by either drug alone, with the maximum protection afforded being 100%. Pretreatment with 2-PAM (10(-6)M) plus physostigmine or pyridostigmine was ineffective. These results suggest that the combinations of atropine plus 2-PAM or physostigmine are effective in the prevention of the lethal effects induced by DFP in A. salina larvae.

The combination of huperzine A and imidazenil is an effective strategy to prevent diisopropyl fluorophosphate toxicity in mice.

Diisopropyl fluorophosphate (DFP) causes neurotoxicity related to an irreversible inhibition of acetylcholinesterase (AChE). Management of this intoxication includes: (i) pretreatment with reversible blockers of AChE, (ii) blockade of muscarinic receptors with atropine, and (iii) facilitation of GABA(A) receptor signal transduction by benzodiazepines. The major disadvantage associated with this treatment combination is that it must to be repeated frequently and, in some cases, protractedly. Also, the use of diazepam (DZP) and congeners includes unwanted side effects, including sedation, amnesia, cardiorespiratory depression, and anticonvulsive tolerance. To avoid these treatment complications but safely protect against DFP-induced seizures and other CNS toxicity, we adopted the strategy of administering mice with (i) small doses of huperzine A (HUP), a reversible and long-lasting (half-life approximately 5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for alpha(5)-containing GABA(A) receptors. Coadministration of HUP (50 microg/kg s.c., 15 min before DFP) with IMI (2 mg/kg s.c., 30 min before DFP) prevents DFP-induced convulsions and the associated neuronal damage and mortality, allowing complete recovery within 18-24 h. In HUP-pretreated mice, the ED(50) of IMI to block DFP-induced mortality is approximately 10 times lower than that of DZP and is devoid of sedation. Our data show that a combination of HUP with IMI is a prophylactic, potent, and safe therapeutic strategy to overcome DFP toxicity.

Histone acetylase inhibitor trichostatin A induces acetylcholinesterase expression and protects against organophosphate exposure.

The biological effects of organophosphorous (OP) chemical warfare nerve agents (CWNAs) are exerted by inhibition of acetylcholinesterase (AChE), which prevents the hydrolysis of the neurotransmitter acetylcholine, leading to hypercholinergy, seizures/status epilepticus, respiratory/cardiovascular failure, and potentially death. Current investigations show that bioscavenger therapy using purified fetal bovine AChE in rodents and non-human primates and the more recently tested human butyrylcholinesterase, is a promising treatment for protection against multiple LD(50) CWNA exposures. Potential impediments, due to the complex structure of the enzyme, purification effort, resources, and cost have necessitated alternative approaches. Therefore, we investigated the effects of transcriptional inducers to enhance the expression of AChE to achieve sufficient protection against OP poisoning. Trichostatin A (TSA), an inhibitor of histone deacetylase that de-condenses the chromatin, thereby increasing the binding of transcription factors and mRNA synthesis, was evaluated for induction of AChE expression in various neuronal cell lines. Dose-response curves showed that a concentration of 333 nM TSA was optimal in inducing AChE expression. In Neuro-2A cells, TSA at 333 nM increased the extracellular AChE activity approximately 3-4 fold and intracellular enzyme activity 10-fold. Correlating with the AChE induction, TSA pre-treatment significantly protected the cells against exposure to the organophosphate diisopropylfluorophosphate, a surrogate for the chemical warfare agents soman and sarin. These studies indicate that transcriptional inducers such as TSA up-regulate AChE, which then can bioscavenge any organophosphates present, thereby protecting the cells from OP-induced cytotoxicity. In conclusion, transcriptional inducers are prospective new methods to protect against CWNA exposure.

Anticholinesterase (DFP) toxicity antagonism by chronic donepezil: a potential nerve agent treatment.

Animal studies exploring the antagonism of irreversible cholinesterase inhibitors (i.e. nerve agents) such as soman and sarin have shown that pretreatment with the reversible centrally acting cholinesterase inhibitor, physostigmine, alone or in conjunction with the centrally acting anticholinergic drug, scopolamine, antagonizes the lethality and toxicity of these agents. This study evaluated the effects of pretreatment with the oral cholinesterase inhibitor and anti-Alzheimer's agent, donepezil (Aricept) on the hypokinetic, hypothermic and diarrhea-inducing effects of the irreversible long-acting cholinesterase inhibitor, diisopropylfluorophosphate (DFP) in adult Sprague-Dawley rats. Donepezil (2 mg/kg), given acutely (30 min pretreatment) or chronically (10 daily treatments), significantly antagonized the hypothermia, hypoactivity and diarrhea induced by DFP (1.25 mg/kg) administration. The effects were most prominent 4 and 6 h after the injection of DFP and some protection was observed even when the last treatment of the chronic donepezil protocol was given 24 h before the DFP injection. Although these phenomena are not the same as lethality, they may be parallel phenomena, and our results may have therapeutic implications for the treatment of nerve agent toxicity.

Organophosphate-induced convulsions and prevention of neuropathological damages.

Such organophosphorus (OP) compounds as diisopropylfluorophosphate (DFP), sarin and soman are potent inhibitors of acetylcholinesterases (AChEs) and butyrylcholinesterases (BChEs). The acute toxicity of OPs is the result of their irreversible binding with AChEs in the central nervous system (CNS), which elevates acetylcholine (ACh) levels. The protective action of subcutaneously (SC) administered antidotes or their combinations in DFP (2.0 mg/kg BW) intoxication was studied in 9-10-weeks-old Han-Wistar male rats. The rats received AChE reactivator pralidoxime-2-chloride (2PAM) (30.0 mg/kg BW), anticonvulsant diazepam (2.0 mg/kg BW), A(1)-adenosine receptor agonist N(6)-cyclopentyl adenosine (CPA) (2.0 mg/kg BW), NMDA-receptor antagonist dizocilpine maleate (+-MK801 hydrogen maleate) (2.0 mg/kg BW) or their combinations with cholinolytic drug atropine sulfate (50.0 mg/kg BW) immediately or 30 min after the single SC injection of DFP. The control rats received atropine sulfate, but also saline and olive oil instead of other antidotes and DFP, respectively. All rats were terminated either 24 h or 3 weeks after the DFP injection. The rats treated with DFP-atropine showed severe typical OP-induced toxicity signs. When CPA, diazepam or 2PAM was given immediately after DFP-atropine, these treatments prevented, delayed or shortened the occurrence of serious signs of poisoning. Atropine-MK801 did not offer any additional protection against DFP toxicity. In conclusion, CPA, diazepam and 2PAM in combination with atropine prevented the occurrence of serious signs of poisoning and thus reduced the toxicity of DFP in rat.

Antagonism of anticholinesterase (DFP) toxicity by donepezil plus scopolamine: a preliminary study.

Studies in animals exploring the antagonism of the cholinesterase inhibitors soman and sarin have shown that pretreatment with low doses of the centrally acting cholinesterase inhibitor, physostigmine, alone or in conjunction with the centrally acting anticholinergic agent, scopolamine, is effective against their lethality and toxicity. The current study evaluated the effects of pretreatment with the oral anticholinesterase agent, donepezil (Aricept, 2.0 mg/kg), used to treat Alzheimer's disease, with and without scopolamine in decreasing the hypothermic, hypokinetic, and diarrhea-inducing effects of the irreversible long-acting cholinesterase inhibitor diisopropyl fluorophosphate (DFP, 1.0 mg/kg) in adult Flinders sensitive line (FSL) male rats. Donepezil alone and donepezil plus scopolamine (0.1 mg/kg) to a greater extent antagonized the decrease in temperature, hypoactivity, and induction of diarrhea due to DFP observed at 4 h after its administration. However, donepezil alone induced hypothermia at 1 and 2 h after treatment. Therefore, these preliminary findings are encouraging, but many additional studies are needed to establish the effectiveness of donepezil as a prophylactic agent against irreversible cholinesterase inhibition by DFP.

Long circulating liposomes encapsulating organophosphorus acid anhydrolase in diisopropylfluorophosphate antagonism.

These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.

Spin trapping agent phenyl-N-tert-butylnitrone prevents diisopropylphosphorofluoridate-induced excitotoxicity in skeletal muscle of the rat.

Indirect evidence suggests that reactive oxygen species (ROS) may mediate muscle fiber necrosis following muscle hyperactivity induced by the anticholinesterase diisopropylphosphorofluoridate (DFP). Pronounced muscle fasciculations and muscle fiber necrosis were seen when acetylcholinesterase (AChE) activity was reduced to less than 30% of control. The spin trapping agent phenyl-N-tert-butylnitrone (PBN) was used in vivo to directly assess the formation of ROS during DFP (1.75 mg/kg, s.c.) induced muscle hyperactivity. Pretreatment with PBN (300 mg/kg, i.p.), the concentration necessary for in vivo spin trapping, prevented muscle hyperactivity as well as necrosis and attenuated the DFP induced AChE inhibition otherwise seen in DFP only treated rats. PBN had no effect when given after fasciculations were established. Muscle extracts from PBN and DFP treated rats subjected to electron spin resonance (ESR) spectroscopy tested negative for ROS. While the role of PBN as an antioxidant is well established, its prophylactic effect against excitotoxity induced by an AChE inhibitor are due to its protection of AChE, an unexpected non-antioxidant action.

Novel protein targets for organophosphorus compounds.

Inhibition of tritiated di-isopropyl phosphorofluoridate labelling by a range of organophopshorus compounds was used to screen for novel OP-reactive targets in rat-brain homogenates. Analysis of target proteins was conducted by SDS/PAGE and detection of tritiated proteins using a thin layer chromatography (TLC) linear analyser. Two major sites of 3H-DFP labelling were found with relative molecular masses of 30 and 85 kDa. Rates of reaction of these labelling sites with a range of OP compounds were compared to that of acetylcholinesterase. The 30 kDa band was found to be more sensitive to paraoxon, dichlorvos and diazoxon than acetylcholinesterase. The 85 kDa band was found to be more sensitive to dichlorvos and diazoxon than acetylcholinesterase. Neither labelling band reacted with chlorfenvinphos or demeton-s-methyl at significant rates.

Identification and isolation of a 155-kDa protein with neuropathy target esterase activity.

A method is presented for the isolation of a 155-kDa protein that possesses phenyl valerate hydrolysis activity in the presence of paraoxon but is inhibited by mipafox; the functional definition of neuropathy target esterase (neurotoxic esterase; NTE). Microsomes, isolated from 18-day-old chicken embryos were treated with phospholipase A2 to solubilize the NTE activity. The extract was then combined with polyoxyethylene W1 detergent and resolved by gel filtration chromatography to yield an active fraction with an approximate mass of 200 kDa. This fraction was further purified by preparative isoelectric focusing and native electrophoresis to yield two separate bands possessing NTE activity. The slower migrating band was highly enriched in a 155-kDa protein that was identified as a source of the NTE activity by affinity chromatography using 3-(9'-mercaptononylthio)-1,1,1-trifluoro-propan-2-one bound to Sepharose CL6B. This represents the first report of the isolation of NTE in its active form and aids in the confirmation of the 155-kDa protein as the most likely candidate for NTE.

beta-Eudesmol as an antidote for intoxication from organophosphorus anticholinesterase agents.

beta-Eudesmol, a sesquiterpenol present in Chinese herbs, improved the tetanic contraction impaired by diisopropylfluorophosphate in isolated mouse diaphragm preparations by an inhibition of the regenerative acetylcholine release. The antagonism was enhanced when a small concentration of obidoxime was present. Neither enzyme reactivation nor curare-like action was evident. beta-Eudesmol (300 mg/kg, i.p.) elevated the LD50 of diisopropylfluorophosphate (s.c.) in control mice from 4.2 to 6.4 mg/kg and in mice pretreated with atropine from 7.8 to 10.6 mg/kg. In mice pretreated with atropine and obidoxime, beta-eudesmol showed a greater synergistic effect, increasing the LD50 from 281 to more than 800 mg/kg. beta-Eudesmol also markedly alleviated diisopropylfluorophosphate-induced muscle fasciculation, tremor and convulsion and prolonged the time to death. It is proposed that beta-eudesmol may be added to the standard antidotal regimen (atropine plus obidoxime) for treating organophosphate intoxication.

Use of C1300 neuroblastoma cells to evaluate the protective value of hexamethonium, trimethaphan, hemicholinium, and triethylcholine against diisopropyl phosphorofluoridate toxicity.

Our intent was to evaluate the C1300 neuroblastoma cell as an in vitro system for studying the mode of action and efficacy of drugs used to treat or prevent organophosphate intoxication. The anticholinergic drugs hexamethonium, trimethaphan, and hemocholinium and the triethylcholine and cholinesterase/reactivator 2-pyridine aldoxime methochloride (2-PAM) have been shown to be effective in preventing intoxication by diisopropyl phosphorofluoridate (also known as diisopropyl fluorophosphate, DFP) in vivo. We determined their efficacy in preventing cell death (as measured by trypan blue exclusion) of neuroblastoma cells alone or in combination. We also determined their efficacy in reversing the cytotoxic effects of DFP on cell DNA synthesis (as measured by [3H]-thymidine incorporation), cell RNA synthesis (as measured by [3H]uridine incorporation), and on cell protein synthesis (as measured by [3H]leucine incorporation). The maximal nontoxic doses of the drugs in vitro were determined. All anticholinergic agents studied reduced the cytotoxicity of DFP using one or more parameters. 2-PAM, the cholinesterase reactivator, enhanced the cytotoxicity of DFP on cultured cells at a high concentration (1 mg/mL) and reduced it at a lower concentration (0.3 mg/mL). All four anticholinergic agents were capable of enhancing the uptake of [3H]thymidine. Only hexamethonium and hemicholinium reversed DFP inhibition of DNA synthesis. RNA synthesis was not affected by any anticholinergic agent and no agent reversed DFP inhibition of RNA synthesis. Protein synthesis was enhanced by every anticholinergic agent except hemicholinium; the inhibition of protein synthesis by DFP was reversed by trimethaphan and triethylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase protection and the anti-diisopropylfluorophosphate efficacy of E2020.

The reversible noncovalent inhibitor of acetylcholinesterase (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methylpiperidine hydrochloride (E2020) was shown to inhibit electric eel acetylcholinesterase with high affinity in a mixed competitive-non-competitive way (Ki = 8.2 nM; Ki' = 13 nM). The pretreatment of electric eel acetylcholinesterase with E2020 dose-dependently prevented the inactivation of the enzyme by 40 microM diisopropylfluorophosphate. The EC50 for this protective effect (95% confidence limits) was 85 (76-96) nM, whereas under the same conditions E2020 IC50 was 12.3 (9.6-16) nM. E2020 injected together with atropine sulfate (17.4 mg/kg) into mice at doses in the range of 1.04-6.24 mg/kg 15 min before diisopropylfluorophosphate, caused a dose-dependent increase in diisopropylfluorophosphate LD50, resulting in protection ratios varying from 3.1 to 9.2. The effectiveness of E2020 antidotal effect was inversely correlated to the time between pretreatment and diisopropylfluorophosphate administration, being maximal when E2020 was injected 15 min, and possibly less than 15 min, before poisoning. From these experiments it is concluded that E2020 exerts a protective action against acute diisopropylfluorophosphate-poisoning in the mouse, presumably by protecting acetylcholinesterase from irreversible inactivation by this agent.

Prevention of organophosphate intoxication with presynaptic cholinergic blockers.

In Vivo cardiovascular neuromuscular preparation of rats was used to study the prophylaxis and mechanism of diisopropyl fluorophosphate (DFP) intoxication by presynaptic cholinolytic agents (ganglionic blocking agents), including hexamethonium, trimethaphan and mecamylamine. The lethal action of DFP was partially or completely prevented by pretreatment of rats with hexamethonium (10 mg/kg), trimethaphan (80 mg/kg) and mecamylamine (30 mg/kg). Combined use of these drugs with 2-PAM (100 mg/kg) improved further the prophylaxis of DFP lethality. The mechanism of prophylaxis could be due to the reduction of acetylcholine turnover and accumulation at the synaptic cleft as hemicholinium prevented the DFP intoxication efficiently. In all cases, DFP caused cardiovascular suppression before neuromuscular blockade indicating that the cardiovascular system is much more sensitive than the neuromuscular junction to DFP intoxication.

Selected biological activities of novel selenonium choline analogs.

1. The novel choline analogs selenonium choline (SeCh) and acetylselenonium choline (ASeCh) have been examined for selected biological activities. 2. ASeCh was found to be an alternative substrate for acetylcholine esterase with Km and Vmax values similar to acetylcholine. 3. ASeCh and SeCh inhibited acetylthiocholine hydrolysis by acetylcholinesterase with IC50 values similar to acetylcholine and choline. 4. SeCh exerted a protective action against physostigmine and DFP induced toxicity. 5. SeCh (85 mg/kg) was found to be 3 times more toxic in mice than choline.

In vitro protection of acetylcholinesterase and butyrylcholinesterase by tetrahydroaminoacridine. Comparison with physostigmine.

The protective action of 1,2,3,4-tetrahydro-9-aminoacridine (THA) against the long-lasting inactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) brought about by diisopropylfluorophosphate (DFP) and physostigmine, as well as by neostigmine in the case of AChE only, was evaluated by a dilution technique using Electrophorus electricus AChE and horse serum BuChE as target enzymes. In parallel experiments, the ability of physostigmine itself to protect these enzymes from DFP was evaluated and compared with that of THA. THA pretreatment was seen to prevent in a dose-dependent manner the inhibition of both AChE and BuChE. However, it was appreciably more potent towards AChE than towards BuChE. THA mean EC50 values for protecting AChE against 10, 40 and 100 microM DFP were 0.04, 0.16 and 0.45 microM, respectively; against 1 microM physostigmine the value was 1.8 microM and against 1.2 microM neostigmine it was 3.0 microM. The THA mean EC50 value for protecting BuChE against 3 microM physostigmine was 0.55 microM and the values for protecting against 3, 10 and 40 microM DFP were 1.5, 3 and greater than 10 microM, respectively. The protective action of THA was time independent: recovery of the maximal enzymic activity was immediate upon dilution. Unlike THA, the protective action of physostigmine developed progressively after dilution and was maximal within 3-4 (AChE) or 6-8 hr (BuChE). Under our experimental conditions, 0.3 microM physostigmine protected approximately 70% of AChE from 40 microM DFP and 5 microM physostigmine protected 9 and 47% of BuChE from 40 and 3 microM DFP, respectively. The results of this work suggest that THA exerts its protective action by shielding the active site of AChE and BuChE from the attack of the inactivating agents on account of its higher enzymic affinity, whereas the protective action of physostigmine against DFP takes advantage also of the carbamylation of the enzyme. These results are in line with the hypothesis that protection of AChE is the primary mechanism responsible for the antidotal action of THA against organophosphorus poisoning.