Porphyrins are a family of highly conjugated molecules that strongly absorb visible light and fluoresce intensely. These molecules are sensitive to changes in their immediate environment and have been widely described for optical detection applications. Surfactant-templated organosilicate materials have been described for the semi-selective adsorption of small molecule contaminants. These structures offer high surface areas and large pore volumes within an organized framework. The organic bridging groups in the materials can be altered to provide varied binding characteristics. This effort seeks to utilize the tunable binding selectivity, high surface area, and low materials density of these highly ordered pore networks and to combine them with the unique spectrophotometric properties of porphyrins. In the porphyrin-embedded materials (PEMs), the organosilicate scaffold stabilizes the porphyrin and facilitates optimal orientation of porphyrin and target. The materials can be stored under ambient conditions and offer exceptional shelf-life. Here, we report on the design of PEMs with specificity for organophosphates and compounds of similar structure.

Genetic mechanisms responsible for organophosphate (OP)-induced behavioral changes remain obscure. In the present study, provisional quantitative trait loci (QTL) associated with acute sensitivity or insensitivity to hypolocomotion produced by the OP paraoxon were identified. Naive adult male and female mice of the BXD/Ty series (22 different BXD strains plus C57BL/6J and DBA/2J progenitor strains) received 0 or 0.25 mg/kg paraoxon (IP), immediately before placement in an activity chamber for a 30-min test. As expected, based on dose-response and time course studies with Swiss-Webster, C57BL/6, and DBA/2 mice, paraoxon treatment reduced locomotor activity in most, but not all BXD strains. Heritability (proportion of phenotypic variability attributed to genetic differences) was 0. 58 for the paraoxon treatment effect. Difference scores (strain mean for vehicle activity minus strain mean for paraoxon activity), and percent change in activity of paraoxon-treated mice compared to vehicle-treated mice were calculated for each BXD strain. QTL analyses using activity difference scores and percentage change in activity were conducted using a database with over 1300 unique genetic markers. Several provisional QTL found on different chromosomes were associated with the activity phenotype. Of these, several markers attained p<0.01 or greater. These were as follows: Chr 1: Ly9, p<0.006; Chr 6: D6Ncvs44, p<0.0005; Chr 9: D9Mit15, p<0. 003; Chr 11: D11Ncvs76, p<0.002; Chr 15: Tstap198, p<0.008. In addition, several markers on chromosome 3 approached p<0.01. Identified genes found near these regions include two plasma carboxylesterase alleles on chromosomes 6 and 9, a glutamate receptor subtype on chromosome 11 and a glycine receptor subunit on chromosome 11, raising the possibility that these genes could be the basis for these provisional QTLs.

Organophosphates (OPCs), useful agents as pesticides, also represent a serious health hazard. Standard therapy with atropine and established oxime-type enzyme reactivators is unsatisfactory. Experimental data indicate that superior therapeutic results can be obtained when reversible cholinesterase inhibitors are administered before OPC exposure. Comparing the protective efficacy of five such cholinesterase inhibitors (physostigmine, pyridostigmine, ranitidine, tacrine, or K-27), we observed best protection for the experimental oxime K-27. The present study was undertaken in order to determine if additional administration of K-27 immediately after OPC (paraoxon) exposure can improve the outcome.

In vitro experiments previously published demonstrated the ability of fullerenes to decrease the capability of organophosphorus (OP) compounds to inhibit acetylcholinesterase. Experiments described herein demonstrate molecular level affinity interactions between fullerenes and the OP test compound paraoxon with NMR spectroscopy. The calculated binding constant of 19 M-1 indicates that this binding was not covalent.

Organophosphorus compounds (OP) are highly toxic molecules used as insecticides that inhibit cholinesterase enzymes involved in neuronal transmission. The intensive use of OP for vector control and agriculture has led to environmental pollutions responsible for severe intoxications and putative long-term effects on humans and wild animals. Many in vivo models were studied over the years to assess OP acute toxicity, but the long-term effects are poorly documented. Planarian, a freshwater flatworm having a cholinergic system, has emerged as a new original model for addressing both toxicity and developmental perturbations. We used Schmidtea mediterranea planarians to evaluate long-term effects of paraoxon-ethyl at two sublethal concentrations over three generations. Toxicity, developmental perturbations and disruption of behavior were rapidly observed and higher sensitivity to paraoxon-ethyl of next generations was noticed suggesting that low insecticide doses can induce transgenerational effects. With the view of limiting OP poisoning, SsoPox, an hyperthermostable enzyme issued from the archaea Saccharolobus solfataricus, was used to degrade paraoxon-ethyl prior to planarian exposure. The degradation products, although not lethal to the worms, were found to decrease cholinesterase activities for the last generation of planarians and to induce abnormalities albeit in lower proportion than insecticides.

We investigated the effect of paraoxon on vascular contractility using organ baths in thoracic aortic rings of rabbits and examined the effect of paraoxon on calcium homeostasis using a whole-cell patch-clamp technique in isolated aortic smooth muscle cells of rabbits. The findings show that administration of paraoxon (30 microM) attenuated thoracic aorta contraction induced by phenylephrine (1 microM) and/or a high K+ environment (80 mM) in both the presence and absence of thoracic aortic endothelium. This inhibitory effect of paraoxon on vasoconstrictor-induced contraction was abolished in the absence of extracellular Ca2+, or in the presence of the Ca2+ channel inhibitor, verapamil. But atropine had little effect on the inhibitory effect of paraoxon on phenylephrine-induced contraction. Paraoxon also attenuated vascular smooth muscle contraction induced by the cumulative addition of CaCl2 and attenuated an increase of intracellular Ca2+ concentration induced by K+ in vascular smooth muscle cells. Moreover, paraoxon (30 microM) inhibited significantly L-type calcium current in isolated aortic smooth muscle cells of rabbits. In conclusion, our results demonstrate that paraoxon attenuates vasoconstrictor-induced contraction through inhibiting Ca2+ influx in the rabbits thoracic aorta.

One of the major signs of severe organophosphate poisoning is seizures. Previous studies have shown that both muscarinic agonist- and organophosphate-induced seizures require activation of muscarinic acetylcholine receptors in the central nervous system. Seizures induced by the muscarinic agonist pilocarpine require the M1 receptor and are modulated by cannabinoid CB1 receptors. In this study, we determined whether M1 and CB1 receptors also regulated seizures induced by the organophosphate paraoxon. We found no differences in seizures induced by paraoxon in wild-type (WT) and M1 knockout (KO) mice, indicating that in contrast to pilocarpine seizures, M1 receptors are not required for paraoxon seizures. Furthermore, we found that pilocarpine administration resulted in seizure-independent activation of ERK in the hippocampus in a M1 receptor-dependent manner, while paraoxon did not induce seizure-independent activation of ERK in the mouse hippocampus. This shows that pilocarpine and paraoxon activated M1 receptors in the hippocampus to different extents. There were no differences in seizures induced by paraoxon in WT and CB1 KO mice, and neither CB1 agonist nor antagonist administration had significant effects on paraoxon seizures, indicating that, in contrast to pilocarpine seizures, paraoxon seizures are not modulated by CB1 receptors. These results demonstrate that there are fundamental molecular differences in the regulation of seizures induced by pilocarpine and paraoxon.

Synthesis of the acetylcholinesterase inhibitor paraoxon (POX) as a carbon-11 positron emission tomography tracer ([11C]POX) and profiling in live rats is reported. Naïve rats intravenously injected with [11C]POX showed a rapid decrease in parent tracer to ∼1%, with an increase in radiolabeled serum proteins to 87% and red blood cells (RBCs) to 9%. Protein and RBC leveled over 60 minutes, reflecting covalent modification of proteins by [11C]POX. Ex vivo biodistribution and imaging profiles in naïve rats had the highest radioactivity levels in lung followed by heart and kidney, and brain and liver the lowest. Brain radioactivity levels were low but observed immediately after injection and persisted over the 60-minute experiment. This showed for the first time that even low POX exposures (∼200 ng tracer) can rapidly enter brain. Rats given an LD50 dose of nonradioactive paraoxon at the LD50 20 or 60 minutes prior to [11C]POX tracer revealed that protein pools were blocked. Blood radioactivity at 20 minutes was markedly lower than naïve levels due to rapid protein modification by nonradioactive POX; however, by 60 minutes the blood radioactivity returned to near naïve levels. Live rat tissue imaging-derived radioactivity values were 10%-37% of naïve levels in nonradioactive POX pretreated rats at 20 minutes, but by 60 minutes the area under the curve (AUC) values had recovered to 25%-80% of naïve. The live rat imaging supported blockade by nonradioactive POX pretreatment at 20 minutes and recovery of proteins by 60 minutes. SIGNIFICANCE STATEMENT: Paraoxon (POX) is an organophosphorus (OP) compound and a powerful prototype and substitute for OP chemical warfare agents (CWAs) such as sarin, VX, etc. To study the distribution and penetration of POX into the central nervous system (CNS) and other tissues, a positron emission tomography (PET) tracer analog, carbon-11-labeled paraoxon ([11C]POX), was prepared. Blood and tissue radioactivity levels in live rats demonstrated immediate penetration into the CNS and persistent radioactivity levels in tissues indicative of covalent target modification.

The chemical structure of organophosphate compounds (OPs) is a well-known factor which modifies the acute toxicity of these compounds. We compared ventilation at rest and cholinesterase activities in male Sprague-Dawley rats poisoned with dimethyl paraoxon (DMPO) and diethyl paraoxon (DEPO) at a subcutaneous dose corresponding to 50% of the median lethal dose (MLD). Ventilation at rest was recorded by whole body plethysmography. Total cholinesterase activities were determined by radiometric assay. Both organophosphates decreased significantly the respiratory rate, resulting from an increase in expiratory time. Dimethyl-induced respiratory toxicity spontaneously reversed within 120 min post-injection. Diethyl-induced respiratory toxicity was long-lasting, more than 180 min post-injection. Both organophosphates decreased cholinesterase activities from 10 to 180 min post-injection with the same degree of inhibition of total cholinesterase within an onset at the same times after injection. There were no significant differences in residual cholinesterase activities between dimethyl and diethyl paraoxon groups at any time. The structure of the alkoxy-group is a determinant factor of the late phase of poisoning, conditioning duration of toxicity without significant effects on the magnitude of alteration of respiratory parameters. For same duration and magnitude of cholinesterase inhibition, there was a strong discrepancy in the time-course of effects between the two compounds.

Antidotes against organophosphates often possess physicochemical properties that mitigate their passage across the blood-brain barrier. Cucurbit[7]urils may be successfully used as a drug delivery system for bisquaternary oximes and improve central nervous system targeting. The main aim of these studies was to elucidate the relationship between cucurbit[7]uril, oxime K027, atropine, and paraoxon to define potential risks or advantages of this delivery system in a complex in vivo system. For this reason, in silico (molecular docking combined with umbrella sampling simulation) and in vivo (UHPLC-pharmacokinetics, toxicokinetics; acetylcholinesterase reactivation and functional observatory battery) methods were used. Based on our results, cucurbit[7]urils affect multiple factors in organophosphates poisoning and its therapy by (i) scavenging paraoxon and preventing free fraction of this toxin from entering the brain, (ii) enhancing the availability of atropine in the central nervous system and by (iii) increasing oxime passage into the brain. In conclusion, using cucurbit[7]urils with oximes might positively impact the overall treatment effectiveness and the benefits can outweigh the potential risks.

Organophosphate (OP) pesticides are widely used in the agricultural field and in the prevention of pest infestation in private and public areas of cities. Despite their unquestionable utility, several of these compounds demonstrate toxic effects to the environment and human health. In particular, the occurrence of some organophosphate pesticides is correlated to the incidence of nervous system disorders, especially in children. The detection of pesticide residues in the human body represents an important task to preserve human health. In our work we propose the use of esterase-based biosensors as a viable alternative to the expensive and time-consuming systems currently used for their detection in human fluids. Using the esterase-2 activity, coupled with a fluorescence inhibition assay, we are able to detect very low concentration levels of diethyl (4-nitrophenyl) phosphate (paraoxon) in the range of the femtomole (fmol). Method robustness tests indicate the stability of esterase-2 in a diluted solution of 4% human urine, and we are able to accurately determine concentration levels of paraoxon in the range from 0.1 to 2 picomoles (pmol). The system sensitivity for OP detection is calculated at 524 ± 14.15 fmol of paraoxon recognized at 10% of inhibition, with an estimated limit of quantification of 262 ± 8.12 pmol mL-1. These values are comparable with the most recent analysis methods based on mass spectrometry carried out on human samples for pesticide detection. This research represents a starting point to develop cheap and fast testing methods for a rapid screening of toxic substances in human samples.

Nerve agents and oxon forms of organophosphorus pesticides act as strong irreversible inhibitors of two cholinesterases in the human body: acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8), and are therefore highly toxic compounds. For the recovery of inhibited AChE, antidotes from the group of pyridinium or bispyridinium aldoxime reactivators (pralidoxime, obidoxime, HI-6) are used in combination with anticholinergics and anticonvulsives. Therapeutic efficacy of reactivators (called “oximes”) depends on their chemical structure and also the type of organophosphorus inhibitor. Three novel oximes (K131, K142, K153) with an oxime group in position four of the pyridinium ring were designed and then tested for their potency to reactivate human (Homo sapiens sapiens) AChE (HssACHE) and BChE (HssBChE) inhibited by the pesticide paraoxon (diethyl 4-nitrophenyl phosphate). According to the obtained results, none of the prepared oximes were able to satisfactorily reactivate paraoxon-inhibited cholinesterases. On the contrary, extraordinary activity of obidoxime in the case of paraoxon-inhibited HssAChE reactivation was confirmed. Additional docking studies pointed to possible explanations for these results.

Casualties caused by organophosphorus pesticides are a burden for health systems in developing and poor countries. Such compounds are potent acetylcholinesterase irreversible inhibitors, and share the toxic profile with nerve agents. Pyridinium oximes are the only clinically available antidotes against poisoning by these substances, but their poor penetration into the blood-brain barrier hampers the efficient enzyme reactivation at the central nervous system. In searching for structural factors that may be explored in future SAR studies, we evaluated neutral aryloximes as reactivators for paraoxon-inhibited Electrophorus eel acetylcholinesterase. Our findings may result into lead compounds, useful for development of more active compounds for emergencies and supportive care.

New uncharged conjugates of 6-methyluracil derivatives with imidazole-2-aldoxime and 1,2,4-triazole-3-hydroxamic acid units were synthesized and studied as reactivators of organophosphate-inhibited cholinesterase. Using paraoxon (POX) as a model organophosphate, it was shown that 6-methyluracil derivatives linked with hydroxamic acid are able to reactivate POX-inhibited human acetylcholinesterase (AChE) in vitro. The reactivating efficacy of one compound (5b) is lower than that of pyridinium-2-aldoxime (2-PAM). Meanwhile, unlike 2-PAM, in vivo study showed that the lead compound 5b is able: (1) to reactivate POX-inhibited AChE in the brain; (2) to decrease death of neurons and, (3) to prevent memory impairment in rat model of POX-induced neurodegeneration.

There are a great number of reports with assertions that oxidative stress is produced by organophosphorus compound (OPC) poisoning and is a cofactor of mortality and morbidity in OPC toxicity. In addition, antioxidants have been suggested as adjuncts to standard therapy. However, there is no substantial evidence for the benefit of the use of antioxidants in survival after acute intoxication of OPCs. The present study was conducted to assess the effectiveness of three non-enzymatic antioxidants (NEAOs), N-acetylcysteine (NAC), glutathione (GSH), and ascorbic acid (AA), in acute intoxication of adult male Wister rats with paraoxon. The efficacy of the antioxidants was estimated as both a pretreatment and a concurrent application along with the standard oxime, pralidoxime (2-PAM). Relative risk of death after 48 hours of application was estimated by Cox regression analysis. The results revealed no benefit of either tested NEAO to the improvement in survival of experimental rats. The application of these antioxidants was found to be deleterious when administered along with pralidoxime compared to the treatment with pralidoxime alone. It has been concluded that the tested non-enzymatic antioxidants are not useful in acute toxicity for improving survival rates. However, the individual toxic dynamics of diversified OPCs should not be overlooked and further studies with different OPCs are suggested.

The present work aimed to compare the small, neutral and monoaromatic oxime, isatin-3-oxime (isatin-O), to the commercial ones, pralidoxime (2-PAM) and obidoxime, in a search for a new potential reactivator for acetylcholinesterase (AChE) inhibited by the pesticide paraoxon (AChE/POX) as well as a novel potential scaffold for further synthetic modifications. The multicriteria decision methods (MCDM) allowed the identification of the best docking poses of those molecules inside AChE/POX for further molecular dynamic (MD) studies, while Ellman's modified method enabled in vitro inhibition and reactivation assays. In corroboration with the theoretical studies, our experimental results showed that isatin-O have a reactivation potential capable of overcoming 2-PAM at the initial moments of the assay. Despite not achieving better results than obidoxime, this molecule is promising for being an active neutral oxime with capacity of crossing the blood⁻brain barrier (BBB), to reactivate AChE/POX inside the central and peripheral nervous systems. Moreover, the fact that isatin-O can also act as anticonvulsant makes this molecule a possible multipotent reactivator. Besides, the MCDM method showed to be an accurate method for the selection of the best docking poses generated in the docking studies.

Organophosphates (OPs) are widely used as pesticides and have been employed as warfare agents. OPs inhibit acetylcholinesterase, leading to over-stimulation of cholinergic synapses and can cause status epilepticus (SE). OPs poisoning can result in irreversible brain damage and death. Despite termination of SE, recurrent seizures and abnormal brain activity remain common sequelae often associated with long-term neural damage and cognitive dysfunction. Therefore, early treatment for prevention of seizures is of high interest. Using a rat model of paraoxon poisoning, we tested the efficacy of different neuroprotective and anti-epileptic drugs (AEDs) in suppressing early seizures and preventing brain damage. Electrocorticographic recordings were performed prior, during and after injection of 4.5 LD50 paraoxon, followed by injections of atropine and toxogonin (obidoxime) to prevent death. Thirty minutes later, rats were injected with midazolam alone or in combination with different AEDs (lorazepam, valproic acid, phenytoin) or neuroprotective drugs (losartan, isoflurane). Outcome measures included SE duration, early seizures frequency and epileptiform activity duration in the first 24 -hs after poisoning. To assess delayed brain damage, we performed T2-weighted magnetic resonance imaging one month after poisoning. SE duration and the number of recurrent seizures were not affected by the addition of any of the drugs tested. Delayed brain injury was most prominent in the septum, striatum, amygdala and piriform network. Only isoflurane anesthesia significantly reduced brain damage. We show that acute treatment with isoflurane, but not AEDs, reduces brain damage following SE. This may offer a new therapeutic approach for exposed individuals.

Oximes are used in addition to atropine to treat organophosphate poisoning. However, the efficiency of oximes is still a matter of debate. In vitro experiments suggested than new oximes are more potent than the commercial oximes. However, the antidotal activity of new oximes has not been assessed in vivo.

The organophosphorous hydrolase (PTE) from Brevundimonas diminuta is capable of degrading extremely toxic organophosphorous compounds with a high catalytic turnover and broad substrate specificity. Although the natural substrate for PTE is unknown, its loop remodeling (loop 7-2/H254R) led to the emergence of a homoserine lactonase (HSL) activity that is undetectable in PTE (kcat/km values of up to 2 × 10(4)), with only a minor decrease in PTE paraoxonase activity. In this study, homology modeling and molecular dynamics simulations have been undertaken seeking to explain the reason for the substrate specificity for the wild-type and the loop 7-2/H254R variant. The cavity volume estimated results showed that the active pocket of the variant was almost two fold larger than that of the wild-type (WT) enzyme. pKa calculations for the enzyme (the WT and the variant) showed a significant pKa shift from WT standard values (ΔpKa = 3.5 units) for the His254 residue (in the Arg254 variant). Molecular dynamics simulations indicated that the displacement of loops 6 and 7 over the active site in loop 7-2/H254R variant is useful for N-acyl-L-homoserine lactone (C4-HSL) with a large aliphatic chain to site in the channels easily. Thence the expanding of the active pocket is beneficial to C4-HSL binding and has a little effect on paraoxon binding. Our results provide a new theoretical contribution of loop remodeling to the rapid divergence of new enzyme functions.

Historically, only few chemicals have been identified as neurodevelopmental toxicants, however, concern remains, and has recently increased, based upon the association between chemical exposures and increased developmental disorders. Diminution in motor speed and latency has been reported in preschool children from agricultural communities. Organophosphorus compounds (OPs) are pesticides due to their acute insecticidal effects mediated by the inhibition of acetylcholinesterase, although other esterases as neuropathy target esterase (NTE) can also be inhibited. Other neurological and neurodevelopmental toxic effects with unknown targets have been reported after chronic exposure to OPs in vivo. We studied the initial stages of retinoic acid acid-triggered differentiation of pluripotent cells towards neural progenitors derived from human embryonal carcinoma stem cells to determine if neuropathic OP, mipafox, and non-neuropathic OP, paraoxon, are able to alter differentiation of neural precursor cells in vitro. Exposure to 1 µM paraoxon (non-cytotoxic concentrations) altered the expression of different genes involved in signaling pathways related to chromatin assembly and nucleosome integrity. Conversely, exposure to 5 µM mipafox, a known inhibitor of NTE activity, showed no significant changes on gene expression. We conclude that 1 µM paraoxon could affect the initial stage of in vitro neurodifferentiation possibly due to a teratogenic effect, while the absence of transcriptional alterations by mipafox exposure did not allow us to conclude a possible effect on neurodifferentiation pathways at the tested concentration.