Pharmacological blockade of the calcium plateau provides neuroprotection following organophosphate paraoxon induced status epilepticus in rats.

Organophosphate (OP) compounds which include nerve agents and pesticides are considered chemical threat agents. Currently approved antidotes are crucial in limiting OP mediated acute mortality. However, survivors of lethal OP exposure exhibit delayed neuronal injury and chronic behavioral morbidities. In this study, we investigated neuroprotective capabilities of dantrolene and carisbamate in a rat survival model of paraoxon (POX) induced status epilepticus (SE). Significant elevations in hippocampal calcium levels were observed 48-h post POX SE survival, and treatment with dantrolene (10mg/kg, i.m.) and carisbamate (90mg/kg, i.m.) lowered these protracted calcium elevations. POX SE induced delayed neuronal injury as characterized by Fluoro Jade C labeling was observed in critical brain areas including the dentate gyrus, parietal cortex, amygdala, and thalamus. Dantrolene and carisbamate treatment provided significant neuroprotection against delayed neuronal damage in these brain regions when administered one-hour after POX-SE. These results indicate that dantrolene or carisbamate could be effective adjuvant therapies to the existing countermeasures to reduce neuronal injury and behavioral morbidities post OP SE survival.

Identification of polymorphisms in Cyrtorhinus lividipennis RDL subunit contributing to fipronil sensitivity.

As one of the most important predatory enemies, the miridbug, Cyrtorhinus lividipennis, plays an important role in rice planthoppers control, such as Nilaparvata lugens (brown planthopper). In order to compare insecticide selectivity between C. lividipennis and N. lugens, the contact acute toxicities of six insecticides (diazoxon, paraoxon, carbaryl, fenobucarb, fipronil and ethofenprox) were monitored. The results showed that all tested insecticides were more toxic to C. lividipennis than to N. lugens and fipronil had the biggest difference. The RDL subunit (Cl-RDL) was cloned from C. lividipennis and a RDL isoform (Cl-RDL-In) was also found with 31 amino acids insertion in RDL intracellular region. In order to understand the role of the insertion on insecticide sensitivities, three subunits (Nl-RDL, Cl-RDL and Cl-RDL-In) were constructed to obtain the functional receptors in Xenopus oocytes and the fipronil sensitivities were detected by the voltage-clamp technique. Nl-RDL (IC50=32.36 ± 4.07 µM) was more insensitive to fipronil than Cl-RDL (IC50=6.47 ± 1.12 µM). The insertion in Cl-RDL significantly reduced fipronil sensitivity with IC50 value in Cl-RDL-In of 16.83 ± 2.30 µM. Interestingly, after the elution of fipronil, the current response of Cl-RDL-In appeared obvious recovery, which were not observed in Cl-RDL and Nl-RDL. It might imply that the insertion played a special role in fipronil sensitivity.

Mechanism of interaction of novel uncharged, centrally active reactivators with OP-hAChE conjugates.

A library of more than 200 novel uncharged oxime reactivators was used to select and refine lead reactivators of human acetylcholinesterase (hAChE) covalently conjugated with sarin, cyclosarin, VX, paraoxon and tabun. N-substituted 2-hydroxyiminoacetamido alkylamines were identified as best reactivators and reactivation kinetics of the lead oximes, RS41A and RS194B, were analyzed in detail. Compared to reference pyridinium reactivators, 2PAM and MMB4, molecular recognition of RS41A reflected in its Kox constant was compromised by an order of magnitude on average for different OP-hAChE conjugates, without significant differences in the first order maximal phosphorylation rate constant k(2). Systematic structural modifications of the RS41A lead resulted in several-fold improvement with reactivator, RS194B. Kinetic analysis indicated K(ox) reduction for RS194B as the main kinetic constant leading to efficient reactivation. Subtle structural modifications of RS194B were used to identify essential determinants for efficient reactivation. Computational molecular modeling of RS41A and RS194B interactions with VX inhibited hAChE, bound reversibly in Michaelis type complex and covalently in the pentacoordinate reaction intermediate suggests that the faster reactivation reaction is a consequence of a tighter RS194B interactions with hAChE peripheral site (PAS) residues, in particular with D74, resulting in lower interaction energies for formation of both the binding and reactivation states. Desirable in vitro reactivation properties of RS194B, when coupled with its in vivo pharmacokinetics and disposition in the body, reveal the potential of this oxime design as promising centrally and peripherally active antidotes for OP toxicity.

Identification and characterization of novel catalytic bioscavengers of organophosphorus nerve agents.

In an effort to discover novel catalytic bioscavengers of organophosphorus (OP) nerve agents, cell lysates from a diverse set of bacterial strains were screened for their capacity to hydrolyze the OP nerve agents VX, VR, and soman (GD). The library of bacterial strains was identified using both random and rational approaches. Specifically, two representative strains from eight categories of extremophiles were chosen at random. For the rational approach, the protein sequence of organophosphorus hydrolase (OPH) from Brevundimonas diminuta was searched against a non-redundant protein database using the Basic Local Alignment Search Tool to find regions of local similarity between sequences. Over 15 protein sequences with significant sequence similarity to OPH were identified from a variety of bacterial strains. Some of these matches were based on predicted protein structures derived from bacterial genome sequences rather than from bona fide proteins isolated from bacteria. Of the 25 strains selected for nerve agent testing, three bacterial strains had measurable levels of OP hydrolase activity. These strains are Ammoniphilus oxalaticus, Haloarcula sp., and Micromonospora aurantiaca. Lysates from A. oxalaticus had detectable hydrolysis of VR; Haloarcula sp. had appreciable hydrolysis of VX and VR, whereas lysates from M. aurantiaca had detectable hydrolysis of VR and GD.

In vitro investigation of efficacy of new reactivators on OPC inhibited rat brain acetylcholinesterase.

Organophosphorus compounds (OPC) were developed as warfare nerve agents. They are also widely used as pesticides. The drug therapy of intoxication with OPC includes mainly combination of cholinesterase (ChE) reactivators and cholinolytics. There is no single ChE reactivator having an ability to reactivate sufficiently the inhibited enzyme due to the high variability of chemical structure of the inhibitors. The difficulties in reactivation of ChE activity and slight antidote effect regarding intoxication with some OPC are some of the reasons for continuous efforts to obtain new reactivators of ChE. The aim of the present study was to evaluate the efficacy of some ChE reactivators against OPC intoxication (tabun, paraoxon and dichlorvos) in in vitro experiments and to compare their activity to that known for some currently used oximes (obidoxime, HI-6, 2-PAM). Experiments were carried out using rat brain acetylcholinesterase (AChE). Reactivators showed different activity in the reactivation of rat brain AChE after dichlorvos, paraoxon and tabun inhibition. AChE was easier reactivated after paraoxon treatment. The best effect showed BT-07-4M, obidoxime, TMB-4 and BT-08 from the group of symmetric oximes, and Toxidin, BT-05 and BT-03 from asymmetric compounds. The reactivation of brain AChE inhibited with tabun demonstrated better activity of new compound BT-07-4M, TMB-4 and obidoxime from symmetric oximes, and BT-05 and BT-03 possessing asymmetric structure. All compounds showed low activity toward inhibition of AChE caused by dichlorvos. Comparison of two main structure types (symmetric/asymmetric) showed that the symmetric compounds reactivated better AChE, inhibited with this OPC, than asymmetric ones.

Evaluation of in vitro tests to assess the efficacy of formulations as topical skin protectants against organophosphorus compounds.

Prevention of exposure to the neurotoxic organophosphorus compounds (OP) is a major concern both for pesticide users and soldiers. Skin barrier creams are being developed to complement or replace uncomfortable chemical protective suits. Quick evaluation of formulations efficacy mainly relies on in vitro tests which lead to consistent, complementary and relevant results. The objectives of this work were to determine the consistency of results from in vitro tests and importance of the formulation composition in the skin protective efficacy. The efficacy of three formulations, i.e. oil-in-water and water-in-oil emulsions and perfluorinated compounds-based cream, was evaluated against the OP paraoxon in vitro. Our results indicated that the least effective formulations could be quickly identified by performing in vitro permeation tests with silicone membrane and by evaluating interfacial interactions between formulations and OP. Among the tested formulations, the perfluorinated compounds-based cream could have a broader spectrum of efficacy than emulsions against OP and other toxic chemicals.

Protective drugs in acute large-dose exposure to organophosphates: a comparison of metoclopramide and tiapride with pralidoxime in rats.

Weak and reversible inhibitors of cholinesterase(s), when coadministered in excess with a more potent inhibitor such as organophosphates, can act in a protective manner. The benzamide compound, metoclopramide, confers some protection (putatively via this mechanism) for cholinesterases against inhibition by paraoxon both in vitro and in vivo, after chronic small-dose exposure. Tiapride is a related benzamide. In this study, we compared the protection by metoclopramide and tiapride in rats acutely exposed to large doses of paraoxon with the therapeutic "gold standard," pralidoxime. Group 1 received 1 micromol paraoxon (approximately 75% lethal dose), Group 2 received 50 micromol metoclopramide, Group 3 received 50 micromol tiapride, Group 4 received 50 micromol pralidoxime, Group 5 received 1 micromol paraoxon + 50 micromol metoclopramide, Group 6 1 micromol paraoxon + 50 micromol tiapride, and Group 7 1 micromol paraoxon + 50 micromol pralidoxime. All substances were administered intraperitoneally. The animals were monitored for 48 h and mortality was recorded at 30 min, 1, 2, 3, 4, 24, and 48 h. Blood was taken for red blood cell acetylcholinesterase measurements at baseline, 30 min, 24, and 48 h. With the exception of Group 7, in which some late mortality was observed, mortality occurred mainly in the first 30 min after paraoxon administration with minimal changes occurring thereafter. Mortality at 30 min was 0% in the metoclopramide, tiapride, and pralidoxime groups and 73 +/- 20 (paraoxon), 65 +/- 15 (paraoxon + metoclopramide), 38 +/- 14 (paraoxon + tiapride), and 13 +/- 19 (paraoxon + pralidoxime). Mortality at 48 h was 75 +/- 18 (paraoxon), 67 +/- 17 (paraoxon + metoclopramide), 42 +/- 16 (paraoxon + tiapride), and 27 +/- 24 (paraoxon + pralidoxime). Metoclopramide does not significantly influence mortality after acute large-dose paraoxon exposure. Both tiapride and pralidoxime significantly decreased mortality in our model. The protection conferred by tiapride was significantly less than that conferred by pralidoxime at 30 min, but was not significantly different at 24 and 48 h.

In vitro effects of organophosphorus anticholinesterases on muscarinic receptor-mediated inhibition of acetylcholine release in rat striatum.

The aim of the present study was to evaluate the in vitro modulation of muscarinic autoreceptor function by the organophosphorus (OP) anticholinesterases chlorpyrifos oxon, paraoxon, and methyl paraoxon. Acetylcholine (ACh) release was studied by preloading slices from rat striatum with [3H]choline and depolarizing with potassium (20 mM) in perfusion buffer containing hemicholinium-3 (to prevent reuptake of radiolabeled choline). Under these conditions, chlorpyrifos oxon, paraoxon, and methyl paraoxon (0.1-10 microM) all reduced ACh release in a concentration-dependent manner. Addition of the carbamate acetylcholinesterase (AChE) inhibitor physostigmine (20 microM) to the perfusion buffer also decreased ACh release. When physostigmine was present, the three oxons had no additional effect on ACh release. Concentration-dependent inhibition of AChE activity in striatal slices perfused with chlorpyrifos oxon (0.1, 1, and 10 microM) suggested AChE inhibition was responsible for oxon-mediated alterations in ACh release. To differentiate between direct and indirect actions of the OP toxicants on muscarinic autoreceptors, we compared the effects of the oxons on ACh release under two conditions, i.e., tissues were perfused with buffer containing only hemicholinium-3 or with buffer containing hemicholinium-3, physostigmine, and the nonselective muscarinic receptor blocker atropine (100 nM). In the presence of only hemicholinium-3, concentration-dependent inhibition of ACh release was again noted for all oxons, similar to the effects of the muscarinic agonists carbachol and cis-dioxolane. In the presence of physostigmine and atropine, the relative potencies of all agents were markedly reduced. Interestingly, carbachol, cis-dioxolane, paraoxon, and methyl paraoxon all decreased ACh release as before, but chlorpyrifos oxon (100-300 microM) actually increased ACh release. Together, the results suggest that chlorpyrifos oxon, paraoxon, and methyl paraoxon can activate muscarinic autoreceptors indirectly through inhibition of AChE. Both paraoxon and methyl paraoxon also directly activate whereas chlorpyrifos oxon blocks muscarinic autoreceptor function. Qualitative differences in the direct actions of these oxons at this presynaptic regulatory site could contribute to differential toxicity with high-dose exposures.

The effect of high and low dosages of paraoxon in beta-naphthoflavone-treated rats.

Aliesterases (carboxylesterases) are serine esterases that can serve a protective role for the target acetylcholinesterase (AChE) during organophosphorus insecticide intoxication because the former esterases are alternate phosphorylation sites. The levels of aliesterase activity in liver and plasma and AChE activity in brain regions were investigated after the intravenous administration of paraoxon (P = O) into female rats. The rats were pretreated intraperitoneally with beta-napthoflavone (BNF), which decreases hepatic aliesterase activity following a 3 day in vivo treatment, and/or tri-o-tolyl phosphate (TOTP) to inhibit aliesterases. The liver aliesterases were inhibited less by P = O in BNF-treated rats than in control rats, which suggests that either BNF exposure may have resulted in aliesterases that are less sensitive to P = O inhibition or BNF may have altered P = O's availability. The BNF treatment did not seem to alter the degree of inhibition of the brain AChE activity following the low dosage of paraoxon (0.04 mg/kg). However, the brain AChE activity in the P = O/TOTP/BNF-treated rats was lower than that in the P = O/TOTP-treated rats, suggesting that BNF also caused changes in systems affecting the disposition of P = O in addition to the changes in the hepatic aliesterases. At the high dosage of paraoxon (0.12 mg/kg), the AChE and aliesterase activities showed a pattern similar to that of the low dosage. This suggests that the aliesterases, as altered by BNF exposure, even when nearly completely inhibited, did not alter the response of the target enzyme, AChE, and, therefore, the magnitude of the toxic response.

[The antagonism of phosphorus-containing heterocyclics with the anticholinesterase agent Phosphacol (paraoxon)]. / Antagonizm fosforsoderzhashchikh geterotsiklov s antikholinésteraznym sredstvom fosfakolom (paraoksonom).

Effects of 15 derivatives of 2-R-oxo-(tioxo-3,5,5-trimethyl-1,2-oxaphospholan-3-ol (OPh) on lethality and protective action on cholinesterase (ChE) of mice brain and blood serum as a result of phosphacolum (paraoxonum) poisoning were investigated. Oxo and tioxo derivatives of OPh with O-methyl and O-ethyl, but not O-propyl and O-butyl radicals at phosphoric atom prevented the lethality of mice poisoned with phosphacolum and preserved brain and blood ChE of inhibition. Ethylic and phenylic OPh derivatives also prevented the lethality of poisoned animals, although without the protection of brain and blood plasma ChE. Combined application of OPh O-ethylic derivative with atropinum and dipiroxinum significantly potentiated antidotal effect m mice poisoned by phosphacolum.