Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation.

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1-10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs.

Chlorpyrifos- and chlorpyrifos oxon-induced neurite retraction in pre-differentiated N2a cells is associated with transient hyperphosphorylation of neurofilament heavy chain and ERK 1/2.

Chlorpyrifos (CPF) and CPF-oxon (CPO) are known to inhibit neurite outgrowth but little is known about their ability to induce neurite retraction in differentiating neuronal cells. The aims of this study were to determine the ability of these compounds to destabilize neurites and to identify the key molecular events involved. N2a cells were induced to differentiate for 20h before exposure to CPF or CPO for 2-8h. Fixed cell monolayers labeled with carboxyfluorescein succinimidyl ester or immunofluorescently stained with antibodies to tubulin (B512) or phosphorylated neurofilament heavy chain (Ta51) showed time- and concentration-dependent reductions in numbers and length of axon-like processes compared to the control, respectively, retraction of neurites being observed within 2h of exposure by live cell imaging. Neurofilament disruption was also observed in treated cells stained by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (NFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2h exposure and reduced levels of reactivity of the same antibody following 8h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8h exposure. These findings suggest for the first time that organophosphorothionate pesticide-induced neurite retraction in N2a cells is associated with transient increases in NFH phosphorylation and ERK1/2 activation.

Acute phenthoate self-poisoning: a prospective case series.

BACKGROUND: The clinical characteristics following self-poisoning with organophosphorus (OP) insecticides differs according to the insecticide ingested. Phenthoate is a dimethoxy WHO Hazard Class II OP pesticide with limited literature on its clinical characteristics and outcome. We aimed to better understand its clinical characteristics by studying patients with phenthoate self-poisoning in Sri Lanka. METHODS: We conducted a prospective cohort study of patients presenting with phenthoate self-poisoning to eight hospitals in Sri Lanka between 2002 and 2018. Clinical outcomes were recorded for each patient. Blood samples for measuring plasma phenthoate concentration, cholinesterase activity, and response to oximes were available for a very small number of patients recruited to a clinical trial. RESULTS: Two hundred and ninety-two patients who ingested agricultural phenthoate formulations were included in the study. Median time to admission was 3.9 (IQR 2.4 - 6.8) h. Forty-two (14.4%) patients were intubated, mostly (30/37, 81%) within 24 h of ingestion (median time to intubation 7.2 [IQR 2.6-20.9] h). Median duration of intubation was 74.8 (IQR 26.8-232.5) h; the longest duration in a survivor was 592 h. Nineteen died (case fatality 6.5%, 95% CI 4.0-10.0); median time to death was 37 (IQR 16 - 101.7) h. Median plasma phenthoate concentration in patients with samples (n = 81) was 135 (IQR 62.7-356.5) ng/mL (0.42 µmol/mL [0.2 to 1.1 µmol/mL]). Five of six patients receiving pralidoxime chloride 2 g showed an initial increase in AChE and BuChE activity that was not sustained despite an infusion of pralidoxime. CONCLUSION: Phenthoate self-poisoning has a 6.5% case fatality rate. Most patients who experience respiratory failure undergo early intubation; most deaths occurred among those patients who were intubated less than 24 h after ingestion. There was a non-sustained increase in cholinesterase activity with pralidoxime, but further studies are required to analyse the extent to which oximes are clinically effective in phenthoate self-poisoning.

Differential Impact of Severity and Duration of Status Epilepticus, Medical Countermeasures, and a Disease-Modifier, Saracatinib, on Brain Regions in the Rat Diisopropylfluorophosphate Model.

Acute organophosphate (OP) toxicity poses a significant threat to both military and civilian personnel as it can lead to a variety of cholinergic symptoms including the development of status epilepticus (SE). Depending on its severity, SE can lead to a spectrum of neurological changes including neuroinflammation and neurodegeneration. In this study, we determined the impact of SE severity and duration on disease promoting parameters such as gliosis and neurodegeneration and the efficacy of a disease modifier, saracatinib (AZD0530), a Src/Fyn tyrosine kinase inhibitor. Animals were exposed to 4 mg/kg diisopropylfluorophosphate (DFP, s.c.) followed by medical countermeasures. We had five experimental groups: controls (no DFP), animals with no continuous convulsive seizures (CS), animals with ∼20-min continuous CS, 31-60-min continuous CS, and > 60-min continuous CS. These groups were then assessed for astrogliosis, microgliosis, and neurodegeneration 8 days after DFP exposure. The 31-60-min and > 60-min groups, but not ∼20-min group, had significantly upregulated gliosis and neurodegeneration in the hippocampus compared to controls. In the piriform cortex and amygdala, however, all three continuous CS groups had significant upregulation in both gliosis and neurodegeneration. In a separate cohort of animals that had ∼20 and > 60-min of continuous CS, we administered saracatinib for 7 days beginning three hours after DFP. There was bodyweight loss and mortality irrespective of the initial SE severity and duration. However, in survived animals, saracatinib prevented spontaneous recurrent seizures (SRS) during the first week in both severity groups. In the ∼20-min CS group, compared to the vehicle, saracatinib significantly reduced neurodegeneration in the piriform cortex and amygdala. There were no significant differences in the measured parameters between the naïve control and saracatinib on its own (without DFP) groups. Overall, this study demonstrates the differential effects of the initial SE severity and duration on the localization of gliosis and neurodegeneration. We have also demonstrated the disease-modifying potential of saracatinib. However, its' dosing regimen should be optimized based on initial severity and duration of CS during SE to maximize therapeutic effects and minimize toxicity in the DFP model as well as in other OP models such as soman.

Organophosphate toxicity: updates of malathion potential toxic effects in mammals and potential treatments.

Organophosphorus insecticides toxicity is still considered a major global health problem. Malathion is one of the most commonly used organophosphates nowadays, as being considered to possess relatively low toxicity compared with other organophosphates. However, widespread use may lead to excessive exposure from multiple sources. Mechanisms of MAL toxicity include inhibition of acetylcholinesterase enzyme, change of oxidants/antioxidants balance, DNA damage, and facilitation of apoptotic cell damage. Exposure to malathion has been associated with different toxicities that nearly affect every single organ in our bodies, with CNS toxicity being the most well documented. Malathion toxic effects on liver, kidney, testis, ovaries, lung, pancreas, and blood were also reported. Moreover, malathion was considered as a genotoxic and carcinogenic chemical compound. Evidence exists for adverse effects associated with prenatal and postnatal exposure in both animals and humans. This review summarizes the toxic data available about malathion in mammals and discusses new potential therapeutic modalities, with the aim to highlight the importance of increasing awareness about its potential risk and reevaluation of the allowed daily exposure level.

Serum and Plasma Cholinesterase Activity in the Cape Griffon Vulture (Gyps coprotheres).

Vulture (Accipitridae) poisonings are a concern in South Africa, with hundreds of birds dying annually. Although some of these poisonings are accidental, there has been an increase in the number of intentional baiting of poached rhinoceros (Rhinocerotidae) and elephant (Elephantidae) carcasses to kill vultures that alert officials to poaching sites by circling overhead. The primary chemicals implicated are the organophosphorous and carbamate compounds. Although most poisoning events can be identified by dead vultures surrounding the scavenged carcass, weak birds are occasionally found and brought to rehabilitation centers for treatment. The treating veterinarian needs to make an informed decision on the cause of illness or poisoning prior to treatment. We established the reference interval for serum and plasma cholinesterase activity in the Cape Griffon Vulture ( Gyps coprotheres ) as 591.58-1,528.26 U/L, providing a clinical assay for determining potential exposure to cholinesterase-depressing pesticides. Both manual and automated samplers were used with the butyrylthiocholine method. Species reference intervals for both serum and plasma cholinesterase showed good correlation and manual and automated measurements yielded similar results.

Physiologic and metabolic safety of butyrylcholinesterase gene therapy in mice.

In continuing efforts to develop gene transfer of human butyrylcholinesterase (BChE) as therapy for cocaine addiction, we conducted wide-ranging studies of physiological and metabolic safety. For that purpose, mice were given injections of adeno-associated virus (AAV) vector or helper-dependent adenoviral (hdAD) vector encoding human or mouse BChE mutated for optimal cocaine hydrolysis. Age-matched controls received saline or AAV-luciferase control vector. At times when transduced BChE was abundant, physiologic and metabolic parameters in conscious animals were evaluated by non-invasive Echo-MRI and an automated "Comprehensive Laboratory Animal Monitoring System" (CLAMS). Despite high vector doses (up to 10(13) particles per mouse) and high levels of transgene protein in the plasma (∼1500-fold above baseline), the CLAMS apparatus revealed no adverse physiologic or metabolic effects. Likewise, body composition determined by Echo-MRI, and glucose tolerance remained normal. A CLAMS study of vector-treated mice given 40 mg/kg cocaine showed none of the physiologic and metabolic fluctuations exhibited in controls. We conclude that neither the tested vectors nor great excesses of circulating BChE affect general physiology directly, while they protect mice from disturbance by cocaine. Hence, viral gene transfer of BChE appears benign and worth exploring as a therapy for cocaine abuse and possibly other disorders as well.