Engineering Dynamic Surface Peptide Networks on ButyrylcholinesteraseG117H for Enhanced Organophosphosphorus Anticholinesterase Catalysis.

The single residue mutation of butyrylcholinesterase (BChEG117H) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in KM and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.

Molecular and Biocompatibility Characterization of Red Blood Cell Membrane Targeted and Cell-Penetrating-Peptide-Modified Polymeric Nanoparticles.

Red blood cells (RBCs) express a variety of immunomodulatory markers that enable the body to recognize them as self. We have shown that RBC membrane glycophorin A (GPA) receptor can mediate membrane attachment of protein therapeutics. A critical knowledge gap is whether attaching drug-encapsulated nanoparticles (NPs) to GPA and modification with cell-penetrating peptide (CPP) will impact binding, oxygenation, and the induction of cellular stress. The objective of this study was to formulate copolymer-based NPs containing model fluorescent-tagged bovine serum albumin (BSA) with GPA-specific targeting ligands such as ERY1 (ENPs), single-chain variable antibody (scFv TER-119, SNPs), and low-molecular-weight protamine-based CPP (LNPs) and to determine their biocompatibility using a variety of complementary high-throughput in vitro assays. Experiments were conducted by coincubating NPs with RBCs at body temperature, and biocompatibility was evaluated by Raman spectroscopy, hemolysis, complement lysis, and oxidative stress assays. Data suggested that LNPs effectively targeted RBCs, conferring 2-fold greater uptake in RBCs compared to ENPs and SNPs. Raman spectroscopy results indicated no adverse effect of NP attachment or internalization on the oxygenation status of RBCs. Cellular stress markers such as glutathione, malondialdehyde, and catalase were within normal limits, and complement-mediated lysis due to NPs was negligible in RBCs. Under the conditions tested, our data demonstrates that molecular targeting of the RBC membrane is a feasible translational strategy for improving drug pharmacokinetics and that the proposed high-throughput assays can prescreen diverse NPs for preclinical and clinical biocompatibility.

Subacute pyridostigmine exposure increases heart rate recovery and cardiac parasympathetic tone in rats.

Heart rate recovery (HRR) describes the rapid deceleration of heart rate after strenuous exercise and is an indicator of parasympathetic tone. A reduction in parasympathetic tone occurs in patients with congestive heart failure, resulting in prolonged HRR. Acetylcholinesterase inhibitors, such as pyridostigmine, can enhance parasympathetic tone by increasing cholinergic input to the heart. The objective of this study was to develop a rodent model of HRR to test the hypothesis that subacute pyridostigmine administration decreases cholinesterase activity and accelerates HRR in rats. Ten days after implantation of radiotelemetry transmitters, male Sprague Dawley rats were randomized to control (CTL) or treated (PYR; 0.14 mg/mL pyridostigmine in the drinking water, 29 days) groups. Rats were exercised on a treadmill to record HRR, and blood samples were collected on days 0, 7, 14, and 28 of pyridostigmine administration. Total cholinesterase and acetylcholinesterase (AChE) activity in plasma was decreased by 32%-43% and 57%-80%, respectively, in PYR rats on days 7-28, while plasma butyrylcholinesterase activity did not significantly change. AChE activity in red blood cells was markedly reduced by 64%-66%. HRR recorded 1 minute after exercise was higher in the PYR group on days 7, 14 and 28, and on day 7 when HRR was estimated at 3 and 5 minutes. Autonomic tone was evaluated pharmacologically using sequential administration of muscarinic (atropine) and adrenergic (propranolol) blockers. Parasympathetic tone was increased in PYR rats as compared with the CTL group. These data support the study hypothesis that subacute pyridostigmine administration enhances HRR by increasing cardiac parasympathetic tone.

Nanoparticle Attachment to Erythrocyte Via the Glycophorin A Targeted ERY1 Ligand Enhances Binding without Impacting Cellular Function.

PURPOSE: Nanoparticle (NP) attachment to biocompatible secondary carriers such as red blood cell (RBC) can prolong blood residence time of drug molecules and help create next-generation nanotherapeutics. However, little is known about the impact of RBC-targeted NPs on erythrocyte function. METHODS: The objectives of this study were to develop and characterize in vitro a novel poly-L-lysine (PLL) and polyethylene glycol (PEG) copolymer-based NP containing fluorescent-tagged bovine serum albumin (BSA), and conjugated with ERY1, a 12 amino acid peptide with high affinity for the RBC membrane protein glycophorin A (ENP). RESULTS: Confocal and flow cytometry data suggest that ENPs efficiently and irreversibly bind to RBC, with approximately 70% of erythrocytes bound after 24 h in a physiologic flow loop model compared to 10% binding of NPs without ERY1. Under these conditions, synthesized ENPs were not toxic to the RBCs. The rheological parameters at the applied shear. (0-15 Pa) were not influenced by ENP attachment to the RBCs. However, at high concentration, the strong affinity of ENPs to the glycophorin-A reduced the deformability of the RBC. CONCLUSIONS: ENPs can be efficiently attached to the RBCs without adversely affecting cellular function, and this may potentially enhance circulatory half-life of drug molecules.

Comparative effects of parathion and chlorpyrifos on extracellular endocannabinoid levels in rat hippocampus: influence on cholinergic toxicity.

Parathion (PS) and chlorpyrifos (CPF) are organophosphorus insecticides (OPs) that elicit acute toxicity by inhibiting acetylcholinesterase (AChE). Endocannabinoids (eCBs, N-arachidonoylethanolamine, AEA; 2-arachidonoylglycerol, 2AG) can modulate neurotransmission by inhibiting neurotransmitter release. We proposed that differential inhibition of eCB-degrading enzymes (fatty acid amide hydrolase, FAAH, and monoacylglycerol lipase, MAGL) by PS and CPF leads to differences in extracellular eCB levels and toxicity. Microdialysis cannulae were implanted into hippocampus of adult male rats followed by treatment with vehicle (peanut oil, 2 ml/kg, sc), PS (27 mg/kg) or CPF (280 mg/kg) 6-7 days later. Signs of toxicity, AChE, FAAH and MAGL inhibition, and extracellular levels of AEA and 2AG were measured 2 and 4 days later. Signs were noted in PS-treated rats but not in controls or CPF-treated rats. Cholinesterase inhibition was extensive in hippocampus with PS (89-90%) and CPF (78-83%) exposure. FAAH activity was also markedly reduced (88-91%) by both OPs at both time-points. MAGL was inhibited by both OPs but to a lesser degree (35-50%). Increases in extracellular AEA levels were noted after either PS (about 2-fold) or CPF (about 3-fold) while lesser treatment-related 2-AG changes were noted. The cannabinoid CB1 receptor antagonist/inverse agonist AM251 (3mg/kg, ip) had no influence on functional signs after CPF but markedly decreased toxicity in PS-treated rats. The results suggest that extracellular eCBs levels can be markedly elevated by both PS and CPF. CB1-mediated signaling appears to play a role in the acute toxicity of PS but the role of eCBs in CPF toxicity remains unclear.

Comparing Longitudinal Measures of Cholinesterase as Biomarkers for Insecticide Exposure Among Latinx Children in Rural Farmworker and Urban Nonfarmworker Communities in North Carolina.

OBJECTIVE: In a 2-group prospective design, this study compared seasonal cholinesterase levels of Latinx children in rural farmworker families and comparable urban children to assess the impact of environmental exposure to cholinesterase-inhibiting insecticides. METHODS: Quarterly blood samples and passive dosimeter wristbands were collected over 2 years in 8-year-old children (74 rural, 62 urban). Laboratory analysis assessed total cholinesterase, acetylcholinesterase, and butyrylcholinesterase from blood samples, and insecticides from wristbands. RESULTS: In spring and summer, total cholinesterase and acetylcholinesterase levels were depressed in rural children compared with winter and fall. Butyrylcholinesterase was depressed in rural children in fall compared with spring and summer. Adjustment for insecticide exposure did not affect these associations. CONCLUSIONS: Environmental exposures to cholinesterase-inhibiting insecticides have measurable biochemical effects on blood cholinesterases in rural children from farmworker families.

Concentration-dependent effects of chlorpyrifos oxon on peroxisome proliferator-activated receptor signaling in MCF-7 cells.

Chlorpyrifos oxon (CPO) is the active metabolite of the organophosphorus pesticide, chlorpyrifos. CPO is a potent inhibitor of acetylcholinesterase (AChE) and other serine hydrolases including fatty acid amide hydrolase (FAAH). AChE is critical in regulating cholinergic signaling while FAAH catalyzes the inactivation of fatty acid signaling lipids including the endocannabinoid (eCB) N-arachidonylethanolamine (anandamide, AEA) and eCB-like metabolites (e.g., oleoylethanolamide, OEA). AEA and OEA are both peroxisome proliferator-activated receptor (PPAR) agonists that regulate numerous genes involved in lipid metabolism and energy homeostasis. We used the MCF-7 human breast cancer cell line, which expresses AChE, FAAH and PPAR alpha and gamma subtypes, to evaluate the potential effects of CPO on PPAR-related gene expression in an in vitro human cell system. CPO elicited relatively similar concentration-dependent inhibition of both AChE and FAAH. Marked concentration- and time-dependent changes in the expression of four selected PPAR-related genes, LXRα, ACOX1, ABCG2 and AGPAT2, were noted. These findings suggest chlorpyrifos may influence lipid metabolism through blocking the degradation of eCBs or eCB-like metabolites and in turn affecting PPAR receptor activation. The results highlight the potential for non-cholinesterase actions of this common insecticide metabolite through disruption of PPAR signaling including effects on lipid metabolism, immune function and inflammation.

Autonomic denervation with magnetic nanoparticles.

BACKGROUND: prior studies indicated that ablation of the 4 major atrial ganglionated plexi (GP) suppressed atrial fibrillation. METHODS AND RESULTS: superparamagnetic nanoparticles (MNPs) made of Fe(3)O(4) (core), thermoresponsive polymeric hydrogel (shell), and neurotoxic agent (N-isopropylacrylamide monomer [NIPA-M]) were synthesized. In 23 dogs, a right thoracotomy exposed the anterior right GP (ARGP) and inferior right GP (IRGP). The sinus rate and ventricular rate slowing responses to high-frequency stimulation (20 Hz, 0.1 ms) were used as the surrogate for the ARGP and IRGP functions, respectively. In 6 dogs, MNPs carrying 0.4 mg NIPA-M were injected into the ARGP. In 4 other dogs, a cylindrical magnet (2600 G) was placed epicardially on the IRGP. MNPs carrying 0.8 mg NIPA-M were then infused into the circumflex artery supplying the IRGP. The hydrogel shell reliably contracted in vitro at temperatures ≥ 37°C, releasing NIPA-M. MNPs injected into the ARGP suppressed high-frequency stimulation-induced sinus rate slowing response (40 ± 8% at baseline; 21 ± 9% at 2 hours; P=0.006). The lowest voltage of ARGP high-frequency stimulation inducing atrial fibrillation was increased from 5.9 ± 0.8 V (baseline) to 10.2 ± 0.9 V (2 hours; P=0.009). Intracoronary infusion of MNPs suppressed the IRGP but not ARGP function (ventricular rate slowing: 57 ± 8% at baseline, 20 ± 8% at 2 hours; P=0.002; sinus rate slowing: 31 ± 7% at baseline, 33 ± 8 % at 2 hours; P=0.604). Prussian Blue staining revealed MNP aggregates only in the IRGP, not the ARGP. CONCLUSIONS: intravascularly administered MNPs carrying NIPA-M can be magnetically targeted to the IRGP and reduce GP activity presumably by the subsequent release of NIPA-M. This novel targeted drug delivery system can be used intravascularly for targeted autonomic denervation.

Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice.

Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n=5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 µM) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner.

Dose- and time-related effects of acute diisopropylfluorophosphate intoxication on forced swim behavior and sucrose preference in rats.

Acute intoxication by organophosphorus anticholinesterases (OPs) has been associated with depression and other neuropsychiatric disorders. We previously reported that adult male rats treated with diisopropylfluorophosphate (2.5 mg/kg, sc) showed acute cholinergic signs followed by changes (increased immobility/decreased swimming) in the forced swim test (a measure of behavioral despair) for at least one month. This study was conducted to evaluate the further persistence of changes in the forced swim test out to 4 months and to compare responses in a sucrose preference test, a measure of anhedonia. Adult male rats were treated with vehicle (peanut oil, 1 mL/kg, sc) or DFP (2.0, 2.25 or 2.5 mg/kg) followed by sacrifice 4 h later for measurement of OP-sensitive serine hydrolases (cholinesterase [ChE], fatty acid amide hydrolase [FAAH], and monoacylglycerol lipase [MAGL]) in hippocampus. Additional rats were treated similarly and evaluated for functional signs of acute toxicity from 30 min to 6 days, and then motor activity, forced swim behavior and sucrose preference at approximately 1 week, 1 month and 4 months after dosing. All dosages of DFP elicited serine hydrolase inhibition (ChE, 92-96 %; FAAH, 46-63 %; MAGL, 26-33 %). Body weight was reduced in all DFP-treated groups during the first two weeks, and lethality was noted with the higher dosages. Involuntary movements were elicited in all DFP treatment groups during the first week, but both time of onset and rate of recovery were dose-related. There was a significant reduction in ambulation at one week after the highest dosage (2.5 mg/kg), but no other significant locomotor changes were noted. Immobility was increased and swimming was decreased in the forced swim test at all three time-points by 2.25 mg/kg DFP, and at 2 of 3 time-points by the other dosages. While length of water deprivation and time after DFP dosing affected sucrose preference, DFP treatment had no main effect. We conclude that the forced swim test (a measure of behavioral despair/coping mechanism for inescapable stress) is a robust and persistent neurobehavioral consequence of acute DFP intoxication while sucrose preference, a measure of anhedonia and a common symptom of major clinical depression, is not affected.

Reprogramming the rapid clearance of thrombolytics by nanoparticle encapsulation and anchoring to circulating red blood cells.

Rapid clearance of thrombolytics from blood following intravenous injection is a major clinical challenge in cardiovascular medicine. To overcome this barrier, nanoparticle (NP) based drug delivery systems have been reported. Although superior than conventional therapy, a large proportion of the injected NP is still cleared by the reticuloendothelial system. Previously, we and others showed that ex vivo attachment of bioscavengers, thrombolytics, and nanoparticles (NPs) to glycophorin A receptors on red blood cells (RBCs) improved the blood half-life. This is promising, but ex-vivo approaches are cumbersome and challenging to translate clinically. Here, we developed a novel Ter119-polymeric NP containing tissue plasminogen activator for on-demand targeting of GPA receptors in vivo. Upon intravenous injection, the Ter119-NPs achieved remarkable RBC labeling efficiencies (>95%), resulting in marked enhancement of blood residence time of tPA from minutes to several days without any morphological, hematological, and histological complications. Our approach of RBC labeling with the NPs also prevented reticuloendothelial detections and the activations of innate and adaptive immune system. Data suggest that real-time targeting of therapeutics to RBC with NPs can potentially improve outcomes and reduce complications against a variety chronic disease.

Dose-related gene expression changes in forebrain following acute, low-level chlorpyrifos exposure in neonatal rats.

Chlorpyrifos (CPF) is a widely used organophosphorus insecticide (OP) and putative developmental neurotoxicant in humans. The acute toxicity of CPF is elicited by acetylcholinesterase (AChE) inhibition. We characterized dose-related (0.1, 0.5, 1 and 2mg/kg) gene expression profiles and changes in cell signaling pathways 24h following acute CPF exposure in 7-day-old rats. Microarray experiments indicated that approximately 9% of the 44,000 genes were differentially expressed following either one of the four CPF dosages studied (546, 505, 522, and 3,066 genes with 0.1, 0.5, 1.0 and 2.0mg/kg CPF). Genes were grouped according to dose-related expression patterns using K-means clustering while gene networks and canonical pathways were evaluated using Ingenuity Pathway Analysis®. Twenty clusters were identified and differential expression of selected genes was verified by RT-PCR. The four largest clusters (each containing from 276 to 905 genes) constituted over 50% of all differentially expressed genes and exhibited up-regulation following exposure to the highest dosage (2mg/kg CPF). The total number of gene networks affected by CPF also rose sharply with the highest dosage of CPF (18, 16, 18 and 50 with 0.1, 0.5, 1 and 2mg/kg CPF). Forebrain cholinesterase (ChE) activity was significantly reduced (26%) only in the highest dosage group. Based on magnitude of dose-related changes in differentially expressed genes, relative numbers of gene clusters and signaling networks affected, and forebrain ChE inhibition only at 2mg/kg CPF, we focused subsequent analyses on this treatment group. Six canonical pathways were identified that were significantly affected by 2mg/kg CPF (MAPK, oxidative stress, NFΚB, mitochondrial dysfunction, arylhydrocarbon receptor and adrenergic receptor signaling). Evaluation of different cellular functions of the differentially expressed genes suggested changes related to olfactory receptors, cell adhesion/migration, synapse/synaptic transmission and transcription/translation. Nine genes were differentially affected in all four CPF dosing groups. We conclude that the most robust, consistent changes in differential gene expression in neonatal forebrain across a range of acute CPF dosages occurred at an exposure level associated with the classical marker of OP toxicity, AChE inhibition. Disruption of multiple cellular pathways, in particular cell adhesion, may contribute to the developmental neurotoxicity potential of this pesticide.

Effects of Chlorpyrifos on Cholinesterase and Serine Lipase Activities and Lipid Metabolism in Brains of Rainbow Trout (Oncorhynchus mykiss).

Chlorpyrifos is an organophosphorus insecticide that elicits acute toxicity through inhibition of acetylcholinesterase (AChE), leading to acetylcholine accumulation and prolonged stimulation of cholinergic receptors throughout the central and peripheral nervous systems. Previous studies have indicated that neurodevelopment may also be impaired through alternative pathways, including reduction of cyclic adenosine monophosphate (cAMP)-catalyzed downstream events. The upstream initiating events that underlie noncholinergic neurological actions of chlorpyrifos and other organophosphorus compounds remain unclear. To investigate the potential role of fatty acid signaling disruption as a mechanism of toxicity, lipid metabolism and fatty acid profiles were examined to identify alterations that may play a critical role in upstream signaling in the central nervous system (CNS). Juvenile rainbow trout were treated for 7 days with nominal chlorpyrifos concentrations previously reported to diminish olfactory responses (10, 20, and 40 µg/l). Although lethality was noted higher in doses, measured chlorpyrifos concentrations of 1.38 µg/l (nominal concentration 10 µg/l) significantly reduced the activity of AChE and two serine lipases, monoacylglycerol lipase, and fatty acid amide hydrolase in the brain. Reductions in lysophosphatidylethanolamines (16:0, 18:0, 18:1, and 22:6) derived from the phosphatidylethanolamines and free fatty acids (palmitic acid 16:0, linolenic acid 18:3, eicosadienoic acid 20:2, arachidonic acid 20:4, and docosahexaenoic acid 22:6) were also noted, suggesting that chlorpyrifos inhibited the metabolism of select phospholipid signaling precursors at sublethal concentrations. These results indicate that in addition to AChE inhibition, environmentally relevant chlorpyrifos exposure alters serine lipase activity and lipid metabolites in the trout brain, which may compromise neuronal signaling and impact neurobehavioral responses in aquatic animals.

Pharmacological enhancement of endocannabinoid signaling reduces the cholinergic toxicity of diisopropylfluorophosphate.

Diisopropylfluorophosphate (DFP) elicits cholinergic toxicity by inhibiting acetylcholinesterase, leading to accumulation of the neurotransmitter acetylcholine and excessive stimulation of cholinergic receptors throughout the body. Endocannabinoids inhibit the release of neurotransmitters including acetylcholine via a widely distributed retrograde signaling pathway. Endocannabinoid signaling is therefore a potential therapeutic target for the management of OP poisoning. We first evaluated the relative in vitro and in vivo (2.5mg/kg, sc) effects of DFP on cholinesterase, fatty acid amide hydrolase (FAAH, an endocannabinoid degrading enzyme), monoacylglycerol lipase (MAGL, another endocannabinoid degrading enzyme) and cannabinoid receptor (CB1) binding in rat hippocampus. The effects of WIN 55212-2 (cannabinoid receptor agonist, 1.5mg/kg), URB597 (FAAH inhibitor, 3mg/kg), URB602 (MAGL inhibitor, 10mg/kg) or AM404 (endocannabinoid uptake inhibitor, 10mg/kg) on DFP toxicity were then examined. Adult male rats were given either peanut oil or DFP followed immediately by vehicle or one of the four cannabinomimetic drugs. Functional signs of toxicity were evaluated for 24h and then rats were sacrificed for neurochemical measurements. DFP inhibited cholinesterase, FAAH, MAGL and CB1 receptor binding in vitro in a concentration-dependent manner, with highest and lowest potency against cholinesterase and FAAH, respectively. In vivo, DFP inhibited hippocampal cholinesterase (89%) and FAAH (42%), but had no significant effect on MAGL or CB1 binding. Rats treated with DFP alone showed typical signs of cholinergic toxicity including involuntary movements and excessive secretions (SLUD signs). WIN 55212-2, URB597, URB602 and AM404 all significantly reduced involuntary movements following DFP exposure in a time-dependent manner, and most (URB597, URB602 and AM404) also significantly reduced DFP-induced SLUD signs. These results suggest that enhancing endocannabinoid signaling can attenuate the acute toxicity of DFP and provide rationale for further investigations on the role of endocannabinoids in cholinergic toxicity.

Effects of chlorpyrifos oxon on M2 muscarinic receptor internalization in different cell types.

The muscarinic M2 receptor is a member of the G protein-coupled receptor (GPCR) superfamily. Agonist activation of GPCR leads to their phosphorylation, desensitization, internalization, and subsequent endocytic recycling or lysosomal degradation. Agonist-induced phosphorylation of M2 receptors is mediated by G-protein receptor kinase 2 (GRK2). The active metabolite of the organophosphorus insecticide chlorpyrifos, i.e., chlorpyrifos oxon (CPO), inhibited agonist-induced phosphorylation of human recombinant M2 receptors by GRK2 in vitro in a concentration-dependent manner. In both intact HEL 299 cells (human embryonic lung fibroblasts expressing M2 receptors) and CHO-M2 cells (stably expressing M2 receptors), the muscarinic agonist carbachol (100 microM) led to receptor internalization as determined by reduced specific binding to the membrane-impermeable radioligand [(3)H]-N-methylscopolamine (NMS). CPO alone (100 microM) exerted no significant effect on NMS binding in either HEL 299 or CHO-M2 cells. In HEL 299 cells, CPO did not influence carbachol-induced internalization, whereas in CHO-M2 cells CPO blocked internalization. In primary striatal neurons, M2 receptors appeared widely and diffusely distributed. Exposure to either carbachol or CPO led to apparent receptor internalization with an increased percent of cells exhibiting punctate domains of immunostaining, while combined exposure to both carbachol and CPO led to a significantly higher percent of cells exhibiting this appearance. The data suggest that CPO may differentially influence agonist-stimulated M2 receptor internalization in a cell-dependent manner.

Cholinesterases and the fine line between poison and remedy.

Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) are related enzymes found across the animal kingdom. The critical role of acetylcholinesterase in neurotransmission has been known for almost a century, but a physiological role for butyrylcholinesterase is just now emerging. The cholinesterases have been deliberately targeted for both therapy and toxicity, with cholinesterase inhibitors being used in the clinic for a variety of disorders and conversely for their toxic potential as pesticides and chemical weapons. Non-catalytic functions of the cholinesterases (ChEs) participate in both neurodevelopment and disease. Manipulating either the catalytic activities or the structure of these enzymes can potentially shift the balance between beneficial and adverse effect in a wide number of physiological processes.

Comparative in vitro and in vivo effects of chlorpyrifos oxon in the outbred CD-1 mouse (Mus musculus) and great plains toad (Anaxyrus cognatus).

We compared biochemical, functional, and behavioral responses to the organophosphorus anticholinesterase chlorpyrifos oxon (CPO) in mice (Mus musculus, CD-1) and toads (Anaxyrus cognatus, Great Plains toad). Toads were substantially less sensitive to acute lethality of CPO based on the maximum tolerated (nonlethal) dose (toads, 77 mg/kg; mice, 5.9 mg/kg). Sublethal exposures led to classical signs of toxicity (increased involuntary movements, autonomic secretions) in mice but hypoactivity in toads. Motor performance in an inclined plane test was not affected by CPO in mice but was altered at the highest dosage in toads. Acetylcholinesterase (AChE), butyrylcholinesterase, monoacylglycerol lipase, and fatty acid amide hydrolase activities in brain were inhibited in mice but not in toads, and fatty acid amide hydrolase activity in the liver was inhibited in both species. Toad brain AChE was less sensitive to in vitro inhibition by CPO (50% inhibitory concentration [IC50; 20 min, 37 °C], 101 vs 7.8 nM; IC50 [20 min, 26 °C], 149 vs 6.2 nM), and studies of inhibitor kinetics indicated substantially lower anticholinesterase potency of CPO against the toad brain enzyme. Using an in vitro indirect inhibition assay, preincubation of CPO with toad brain homogenate was more effective than an equivalent mouse brain homogenate at reducing CPO potency. These data suggest that the relatively low sensitivity of toads to cholinergic toxicity is based on the low sensitivity of brain AChE, which in turn may be attributable to more effective target-site detoxification. Environ Toxicol Chem 2018;37:1898-1906. © 2018 SETAC.

Comparative in vivo effects of parathion on striatal acetylcholine accumulation in adult and aged rats.

Aged rats are more sensitive to the acute toxicity of the prototype organophosphate insecticide, parathion. We compared the acute effects of parathion on diaphragm and brain regional cholinesterase activity, muscarinic receptor binding and striatal acetylcholine levels in 3- and 18-month-old male Sprague-Dawley rats. Adult and aged rats were surgically implanted with a microdialysis cannula into the right striatum 5-7 days prior to parathion treatment. Rats were given either vehicle (peanut oil, 2 ml/kg) or one of a range of dosages of parathion (adult: 1.8, 3.4, 6.0, 9.0, 18 and 27 mg/kg, s.c.; aged: 1.8, 3.4, 6 and 9 mg/kg, s.c.) and body weight, functional signs of toxicity, and nocturnal motor activity were recorded for seven days. Three and seven days after parathion treatment, microdialysis samples were collected and rats were subsequently sacrificed for biochemical measurements. Higher dosages of parathion led to significant time-dependent reductions in body weight in both age groups. Rats in both age groups treated with lower dosages showed few overt signs of cholinergic toxicity while equitoxic high dosages (adult, 27 mg/kg; aged, 9 mg/kg) elicited marked signs of cholinergic toxicity (involuntary movements and SLUD [i.e., acronym for Salivation, Lacrimation, Urination and Defecation] signs) with peak effects being noted 3-4 days after treatment. Nocturnal activity (ambulation and rearing) was reduced in both age groups following parathion dosing, with more prominent effects in adults and rearing being more consistently affected. Dose- and time-dependent inhibition of cholinesterase activity was noted in both diaphragm and striatum. Total muscarinic receptor ([(3)H]quinuclidinyl benzilate, QNB) binding was significantly lower in aged rats, and both total binding and muscarinic agonist ([(3)H]oxotremorine methiodide] binding was significantly reduced in both age-groups treated with the highest dosages of parathion (adult, 27 mg/kg; aged, 9 mg/kg). In contrast to relatively similar levels of cholinesterase inhibition, striatal extracellular acetylcholine levels were significantly lower (2.2- to 2.9-fold) in aged rats at both 3 and 7 day time-points compared to adult rats treated with equitoxic dosages (i.e., 9 and 27 mg/kg, respectively). No age-related differences in in vitro striatal acetylcholine synthesis or in vivo acetylcholine accumulation following direct infusion of the cholinesterase inhibitor neostigmine (1 microM) were noted. While aged rats are more sensitive than adults to the acute toxicity of parathion, lesser acetylcholine accumulation was noted in the striatum of aged rats exhibiting similar levels of cholinesterase inhibition. These findings suggest that lesser acetylcholine accumulation may be required to elicit cholinergic signs in the aged rat, possibly based on aging-associated changes in muscarinic receptor density.

Comparative effects of oral chlorpyrifos exposure on cholinesterase activity and muscarinic receptor binding in neonatal and adult rat heart.

Organophosphorus (OP) pesticides elicit acute toxicity by inhibiting acetylcholinesterase (AChE), the enzyme responsible for inactivating acetylcholine (ACh) at cholinergic synapses. A number of OP toxicants have also been reported to interact directly with muscarinic receptors, in particular the M(2) muscarinic subtype. Parasympathetic innervation to the heart primarily regulates cardiac function by activating M(2) receptors in the sinus node, atrial-ventricular node and conducting tissues. Thus, OP insecticides can potentially influence cardiac function in a receptor-mediated manner indirectly by inhibiting acetylcholinesterase and directly by binding to muscarinic M(2) receptors. Young animals are generally more sensitive than adults to the acute toxicity of OP insecticides and age-related differences in potency of direct binding to muscarinic receptors by some OP toxicants have been reported. We thus compared the effects of the common OP insecticide chlorpyrifos (CPF) on functional signs of toxicity and cardiac cholinesterase (ChE) activity and muscarinic receptor binding in neonatal and adult rats. Dosages were based on acute lethality (i.e., 0.5 and 1x LD(10): neonates, 7.5 and 15 mg/kg; adults, 68 and 136 mg/kg). Dose- and time-related changes in body weight and cholinergic signs of toxicity (involuntary movements) were noted in both age groups. With 1x LD(10), relatively similar maximal reductions in ChE activity (95%) and muscarinic receptor binding (approximately 30%) were noted, but receptor binding reductions appeared earlier in adults and were more prolonged in neonates. In vitro inhibition studies indicated that ChE in neonatal tissues was markedly more sensitive to inhibition by the active metabolite of chlorpyrifos (i.e., chlorpyrifos oxon, CPO) than enzyme in adult tissues (IC(50) values: neonates, 17 nM; adults, 200 nM). Chelation of free calcium with EDTA had relatively little effect on in vitro cholinesterase inhibition, suggesting that differential A-esterase activity was not responsible for the age-related difference in cholinesterase sensitivity between age groups. Pre-incubation of neonatal and adult tissues with selective inhibitors of AChE and butyrylcholinesterase (BChE) indicated that a majority (82-90%) of ChE activity in the heart of both neonates and adults was BChE. The rapid onset (by 4h after dosing) of changes in muscarinic receptor binding in adult heart may be a reflection of the more potent direct binding to muscarinic receptors by chlorpyrifos oxon previously reported in adult tissues. The results suggest that ChE activity (primarily BChE) in neonatal heart may be inherently more sensitive to inhibition by some anticholinesterases and that toxicologically significant binding to muscarinic receptors may be possible with acute chlorpyrifos intoxication, potentially contributing to age-related differences in sensitivity.

In vitro and in vivo toxicity of CdTe nanoparticles.

Cadmium telluride (CdTe) nanoparticles exhibit strong and stable fluorescence that is attractive for many applications such as biological probing and solid state lighting. The evaluation of nanoparticle toxicity is important for realizing these practical applications. However, no systematic studies of CdTe nanoparticle toxicity have been reported. We investigated and compared the size- and concentration-dependent cytotoxicity of CdTe nanoparticles in human hepatoma HepG2 cells using the MTT assay. CdTe nanoparticles elicited cytotoxicity in a concentration- and size-dependent manner, with smaller-sized particles exhibiting somewhat higher potency. Lesser cytotoxicity of partially purified CdTe-Red particles (following methanol precipitation and resuspension) suggested that free cadmium ions may contribute to cytotoxicity. We also evaluated the acute toxicity of CdTe-Red particles following intravenous exposure in male rats (2 micromol/kg). Few signs of functional toxicity or clinical (urinary or blood) changes were noted. Interestingly, motor activity was transiently reduced (2 hours after treatment) and then significantly increased at a later timepoint (24 hours after dosing). These studies provide a framework for further characterizing the in vitro and in vivo toxic potential of different types of CdTe nanoparticles and suggest that the nervous system may be targeted by these nanoparticles under some conditions.