The homeostasis of phosphatidylcholine and lysophosphatidylcholine was not disrupted during tri-o-cresyl phosphate-induced delayed neurotoxicity in hens

Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity (OPIDN) except for the essential inhibition of neuropathy target esterase (NTE). We hypothesized that the homeostasis of lysophosphatidylcholine (LPC) and/or phosphatidylcholine (PC) in nervous tissues might be disrupted after exposure to the organophosphates (OP) which participates in the progression of OPIDN because new clues to possible mechanisms of OPIDN have recently been discovered that NTE acts as lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. To bioassay for such phospholipids, we induced OPIDN in hens using tri-o-cresyl phosphate (TOCP) as an inducer with phenylmethylsulfonyl fluoride (PMSF) as a negative control; and the effects on the activities of NTE, LysoPLA and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE enzyme in the brain, spinal cord, and sciatic nerves were examined. The results demonstrated that the activities of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB and PLB were significantly inhibited in both TOCP- and PMSF-treated hens. The inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient in the nervous tissues. Moreover, the NTE inhibited by TOCP was of the aged type, while nearly all of the NTE inhibited by PMSF was of the unaged type. No significant change in PC or LPC levels was observed, while the GPC level was significantly decreased. However, there is no relationship found between the GPC level and the delayed symptoms or aging of NTE. All results suggested that LPC and/or PC homeostasis disruption may not be a mechanism for OPIDN because the PC and LPC homeostasis was not disrupted after exposure to the neuropathic OP, although NTE, LysoPLA, and PLB were significantly inhibited and the GPC level was remarkably decreased.     

Lysophospholipase inhibition by organophosphorus toxicants

Lysophospholipases (LysoPLAs) are a large family of enzymes for removing lysophospholipids from cell membranes. Potent inhibitors are needed to define the importance of LysoPLAs as targets for toxicants and potential therapeutics. This study considers organophosphorus (OP) inhibitors with emphasis on mouse brain total LysoPLA activity relative to the mipafox-sensitive neuropathy target esterase (NTE)-LysoPLA recently established as 17% of the total activity and important in the action of OP delayed toxicants. The most potent inhibitors of total LysoPLA in mouse brain are isopropyl dodecylphosphonofluoridate (also for LysoPLA of Vibrio bacteria), ethyl octylphosphonofluoridate (EOPF), and two alkyl-benzodioxaphosphorin 2-oxides (BDPOs)[(S)-octyl and dodecyl] (IC50 2-8 nM). OP inhibitors acting in vitro and in vivo differentiate a more sensitive portion but not a distinct NTE-LysoPLA compared with total LysoPLA activity. For 10 active inhibitors, NTE-LysoPLA is 17-fold more sensitive than total LysoPLA, but structure-activity comparisons give a good correlation (r(2) = 0.94) of IC50 values, suggesting active site structural similarity or identity. In mice 4 h after intraperitoneal treatment with discriminating doses, EOPF, tribufos (a plant defoliant), and dodecanesulfonyl fluoride inhibit 41-57% of the total brain LysoPLA and 85-99% of the NTE-LysoPLA activity. Total LysoPLA as well as NTE-LysoPLA is decreased in activity in Nte(+/-)-haploinsufficient mice compared to their Nte(+/+) littermates. The lysolecithin level of spinal cord but not brain is elevated significantly following EOPF treatment (3 mg/kg), thereby focusing attention on localized rather than general alterations in lysophospholipid metabolism in OP-induced hyperactivity and toxicity.     

Phenylmethylsulfonyl fluoride protects rats from Mipafox-induced delayed neuropathy

Initiation of organophosphorus-induced delayed neuropathy (OPIDN) is thought to consist of two molecular events involving the phosphorylation of the target enzyme, neurotoxic esterase, or neuropathy target enzyme (NTE), and a subsequent “aging” reaction which transforms the inhibited NTE into a charged moiety critical to the neuropathic process. Compounds that inhibit NTE but cannot age because of their chemical structure abort this two-stage initiation process, and when administered before a neurotoxic organophosphorus compound (OP), protect against the neuropathy by blocking NTE's active site (Johnson, 1970). In support of this, we report that prior exposure to a nonaging NTE inhibitor, phenylmethylsulfonyl fluoride (PMSF), protects rats from neurological damage after subsequent exposure to a neurotoxic OP, Mipafox. Adult, male, Long Evans rats were exposed to either PMSF (250 mg/kg, sc) or to Mipafox (15 mg/kg, ip) and a time course of brain NTE inhibition and recovery was defined. A separate group of PMSF-treated rats was exposed to Mipafox when brain NTE inhibition was 87.7 ± 2.3%. Conversely, another group of rats, pretreated with Mipafox, was dosed with PMSF when NTE inhibition was 90.2 ± 0.8%. A third group of animals, treated with PMSF, was exposed to Mipafox 14 days later, when NTE activity had recovered to within 10 ± 4.2% of control amounts. Histopathological survey (14 to 21 days post-exposure) indicated severe cervical cord damage (damage score ≥3) in the follwing frequencies: PMSF, 0%; Mipafox, 85%; PMSF-4 hr-Mipafox, 0%; Mipafox-4 hr-PMSF, 100%; PMSF-14 days-Mipafox, 75%; controls, 0%. These data indicate that PMSF pretreatment protects rats against Mipafox-induced neurological damage and that the timing of administration and order of presentation are critical to this protection. These results support the hypothesis that the initiation of OPIDN is a multistage event involving inhibition and aging, and that these stages are experimentally separable.     

Phospholipase B activity and organophosphorus compound toxicity in cultured neural cells

Organophosphorus compounds (OP) such as phenyl saligenin phosphate (PSP) and mipafox (MPX) which cause delayed neuropathy, inhibit neuropathy target esterase (NTE), while OPs such as paraoxon (PXN) react more readily with acetylcholinesterase. In yeast and mammalian cell lines, NTE has been shown to have phospholipase B (PLB) activity which deacylates intracellular phosphatidylcholine to glycerophosphocholine (GroPCho) and can be detected by metabolic labeling with [(14)C]choline. Here we investigated PLB activity in primary cultures of mouse neural cells. In cortical and cerebellar granule neurons and astrocytes, [(14)C]GroPCho labeling was inhibited by PSP and MPX: phenyl dipentylphosphinate (PDPP), a non-neuropathic NTE inhibitor, was more potent, while PXN, was substantially less so. In all three cell types, conversion of [(14)C]phosphatidylcholine to [(14)C]GroPCho over 24 h was relatively small (2.3-14%). Consequently, even with >80% inhibition of [(14)C]GroPCho production, increased [(14)C]phosphatidylcholine was not detected. At concentrations of 1-10 microM, only PSP was cytotoxic to cortical and cerebellar granule neurons after 24-h exposure. Moreover, dramatic changes in glial cell morphology were induced by PSP, but not PDPP or MPX, with rapid (2-3 h) rounding up of astrocytes and of Schwann cells in cultures of dissociated mouse dorsal root ganglia. We conclude that PLB activity is present in a variety of cultured mouse neural cell types but that acute loss of this activity is not cytotoxic. Conversely, the rapid toxic effects of PSP in vitro suggest that a serine hydrolase distinct from NTE is required continuously by neurons and glia.     

Cellular function of neuropathy target esterase in lysophosphatidylcholine action

Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.     

Degradation of organophosphorus neurotoxicity in SY5Y neuroblastoma cells by organophosphorus hydrolase (OPH)

Numerous approaches have been studied to degrade organophosphorus (OP) compounds and ameliorate their toxicity. In the current study, the potential of genetically engineered organophosphorus hydrolase (OPH) enzymes to functionally biotransform OP neurotoxicants was examined by assessing effects of OPH-hydrolyzed OPs on acute and delayed indicators of neurotoxicity. SY5Y human neuroblastoma cells were used as a model test system, as these cells respond distinctly to mipafox, which produces OP-induced delayed neuropathy, and paraoxon, which does not. Short-term effects of four OPH-treated OPs on acetylcholinesterase (AChE) and neuropathy target esterase (NTE) activities were measured in retinoic acid-differentiated or undifferentiated cells, and delayed effects of OPH-treated paraoxon or mipafox on levels of neuronal cytoskeletal proteins in nerve growth factor (NGF)-differentiated cells. The anti-AChE activity of paraoxon (maximum 3 muM) and anti-NTE activity of mipafox (250 muM) in SY5Y cells were prevented by biodegradation with OPH. Anti-AChE activities of mipafox, methyl parathion, and demeton-S were partially ameliorated, depending on OP concentration. Intracellular amounts of the 200-kD neurofilament protein NF200 were unchanged after treatment with OPH-treated or buffer-treated paraoxon, as expected, as this endpoint is insensitive to paraoxon. However, NF200 levels rose in cells treated during late differentiation with OPH-treated mipafox. This finding suggests the existence of a threshold concentration of mipafox below which SY5Y cells can maintain their viability for compensating cellular damage due to mipafox in neurite elongation. These results indicate that OPH may be used to biodegrade OPs and remediate their neurotoxic effects in vitro and that AChE and NTE are suitable detectors for OPH amelioration.     

Acetylcholinesterase and neuropathy target esterase activities in 11 cases of symptomatic flight crew members after fume events

ABSTRACT

In modern aviation, so-called fume events such as exposure to an unknown mixture of chemicals introduced into the aircraft cabin with bleed air drawn off at the engines may occur. Human exposure may result in (neuro)toxic symptoms described as so-called “aerotoxic syndrome.” Currently, among other agents organophosphates (OP) are regarded as a likely cause of the observed adverse effects. After fume events 11 flight crew members (9 female/2 male; ages 23–58 yr) were admitted for a medical examination within 5 d post exposure. Individual acetylcholinesterase (AChE) and neuropathy target esterase (NTE) activities were determined. Anamnesis and clinical findings confirmed prominent symptoms of an intoxication, including headache, cognitive difficulties, and neurological disorders, among others. Patient AChE activities ranged from 37 to 50 U/g hemoglobin (reference values: 26.7–50.9 U/g hemoglobin). Ten individuals showed NTE activities ranging from 3.14 to 6.3 nmol phenyl valerate/(min × mg protein) (reference values: 3.01–24), with one patient exhibiting low NTE activity of 1.4. Biochemical effect monitoring was applied to encompass a broad range of AChE-inhibiting compounds such as OP, carbamates, and isocyanates, or to detect inhibition of NTE. The measured AChE activities indicated a subordinate contribution of OP or related compounds to the observed symptoms. All noted NTE activities were clustered at low levels. Our data suggest a likely inhibition of NTE activities in patients after fume events, which warrants further investigation. The observed symptoms may be linked to known chemical compounds in fume events, and it is not possible to infer a direct correlation between manifestations and AChE -inhibiting compounds at this time.     

Adrenergic and cholinergic regulation of phospholipid release in sublingual salivary gland in vitro.

1. The role of adrenergic and cholinergic mediators in the regulation of salivary phospholipid secretion was investigated using rat sublingual acinar cells maintained in the presence of [3H]choline. 2. The release of [3H]choline containing phospholipids was enhanced by beta-adrenergic agonist, isoproterenol to a greater extent than the cholinergic agonist, pilocarpine. 3. The effect of isoproterenol on phospholipid release was observed even after 5 min and by 30 min a 1.7-fold increase in secretion occurred, whereas pilocarpine evoked 1.2-fold increase by 30 min with no discernible effect in 5 min. The isoproterenol effect was blocked by alprenolol, and an inhibition of pilocarpine effect was observed with atropine. 4. In the absence of mediators, 82% of secreted labeled phospholipids were represented by phosphatidylcholine (PC), 4.3% by lysophosphatidylcholine (LPC) and 12.1% by sphingomyeline (Sph), those secreted in the presence of isoproterenol showed significantly lower (37%) content of LPC, while those secreted in response to pilocarpine were substantially richer in LPC (2.5 times) and Sph (27%), and contained less (17%) PC. 5. The results provide first evidence for the involvement of adrenergic and cholinergic mediators in the regulation of salivary phospholipid secretion.     

Chlorpyrifos: Assessment of Potential for Delayed Neurotoxicity by Repeated Dosing in Adult Hens with Monitoring of Brain Acetylcholinesterase, Brain and Lymphocyte Neurotoxic Esterase, and Plasma Butyrylcholinesterase Activities

Previous work has shown that acute exposures to chlorpyrifos(CPS; diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate) cannotproduce >70% inhibition of brain neurotoxic esterase (NTE)and cause organophosphorus compound-induced delayed neurotoxicity(OPIDN) unless the dose is well in excess of the LD50, necessitatingaggressive therapy for cholinergic toxicity. The present studywas carried out to determine if repeated doses of CPS at themaximum tolerated daily dose without prophylaxis against cholinergictoxicity could cause cumulative inhibition of NTE and OPIDN.Adult hens were dosed daily for 20 days with CPS (10 mg/kg/daypo in 2 ml/kg corn oil) or corn oil (vehicle control) (2 ml/kg/daypo) and observed for an additional 4 weeks. Brain acetylcholinesterase(AChE), brain and lymphocyte NTE, and plasma butyrylcholinesterase(BuChE) activities were assayed on Days 0 (control only), 4,10, 15, 20, and 48. During Days 4–20, brain AChE and plasmaBuChE activities from CPS-treated hens were inhibited 58–70%and 49–80% of contemporaneous controls, respectively.At 4 weeks after the end of dosing, brain AChE activity in treatedbirds had recovered to 86% of control and plasma BuChE activitywas 134% of control. Brain and lymphocyte NTE activities oftreated animals throughout the study were 82–99% and 85–128%of control, respectively. Neither brain nor lymphocyte NTE activitiesin treated hens exhibited cumulative inhibition. The 18% inhibitionof brain NTE seen on days 10 and 20 was significant, but substantiallybelow the putative threshold for OPIDN. Body weight of treatedhens decreased 10–25% during Days 4–20 and recoveredto 87% of control by the end of the study. Some treated hensdeveloped a slight staggering gait during the first week ofdosing, which disappeared by the second week. Throughout the4-week observation period, all hens appeared normal and wereable to perch on a horizontal rod. The results indicate thatdaily dosing with CPS at a level sufficient to cause significantloss of body weight as well as marked inhibition of brain AChEand plasma BuChE resulted in no significant change in lymphocyteNTE activity, a maximum inhibition of brain NTE of 18%, no cumulativeinhibition of lymphocyte or brain NTE, and no clinical signsof OPIDN.     

Comparison of chromaffin cells from several animal sources for their use as an in vitro model to study the mechanism of organophosphorous toxicity

It had been observed that the chromaffin cells of bovine adrenal medulla contain high levels of neuropathy target esterase (NTE), the esterase whose inhibition and aging is associated with induction of the organophosphorous induced delayed neuropathy. In this study, total esterase and NTE activities, and their inhibition kinetics by OPs are characterized in adrenal medulla of several species in order to find the best source for chromaffin cells. Total esterase activity in membrane fraction of bovine, equine, porcine, ovine and caprine were 6100+/-840, 4200+/-270, 5000+/-120, 28800+/-3000, and 10800+/-2400mU/gtissue, respectively (mean+/-S.D., n=3-4). NTE represented around 70%, 24%, 58%, 10% and 24% of the total esterases in the same tissues, respectively. It was deduced that NTE represents between 69% and 89% of the "B-activity" (activity resistant to 40microM paraoxon) in the membrane fraction of all species. The mipafox I(50) calculated for 30-min inhibition of NTE at 37 degrees Celsius ranged between 7.4 and 12microM. These values are in the range of that for brain NTE in hen (the usual model for testing OP delayed neurotoxicity). Considering that bovine adrenal medulla contains high NTE activity, that it represents a high proportion of total activity, it is easier to dissect than adrenal medulla from equine, caprine or ovine, and is more readily available than species cited previously, and that its inhibitory properties are similar to the classical hen brain model, it is deduced that bovine adrenal medulla is the most appropriate source of chromaffin cells to study OP toxicity, with porcine as the second alternative. The kinetic properties of chromaffin cell cultures from bovine and porcine were in accordance with their properties in homogenate and subcellular fractions, and they displayed an appropriate stability and viability of the primary culture to be used in in vitro toxicological studies for both mechanistic and testing purposes.     

Biosensor detection of neuropathy target esterase in whole blood as a biomarker of exposure to neuropathic organophosphorus compounds

Neuropathy target esterase (NTE) is the target protein for neuropathic organophosphorus (OP) compounds that produce OP compound-induced delayed neurotoxicity (OPIDN). Inhibition/aging of brain NTE within hours of exposure predicts the potential for development of OPIDN in susceptible animal models. Lymphocyte NTE has also found limited use as a biomarker of human exposure to neuropathic OP compounds. Recently, a highly sensitive biosensor was developed for NTE activity using a tyrosinase carbon-paste electrode for amperometric detection of phenol produced by hydrolysis of the substrate, phenyl valerate. The I50 (20 min at 37 degrees C) for N,N'-di-2-propylphosphorodiamidofluoridate (mipafox) against hen lymphocyte NTE was 6.94 +/- 0.28 microM amperometrically and 6.02 +/- 0.71 microM colorimetrically. For O,O-di1-propyl O-2,2-dichlorvinyl phosphate (PrDChVP), the I50 against hen brain NTE was 39 +/- 8 nM amperometrically and 42 +/- 2 nM colorimetrically. The biosensor enables NTE to be assayed in whole blood, whereas this cannot be done with the usual colorimetric method. Amperometrically, I50 values for PrDChVP against hen and human blood NTE were 66 +/- 3 and 70 +/- 14 nM, respectively. To study the possibility of using blood NTE inhibition as a biochemical marker of neuropathic OP compound exposure, NTE activities in brain and lymphocytes as well in brain and blood were measured 24 h after dosing hens with PrDChVP. Brain, lymphocyte, and blood NTE were inhibited in a dose-responsive manner, and NTE inhibition was highly correlated between brain and lymphocyte (r = .994) and between brain and blood (r = .997). The results suggest that the biosensor NTE assay for whole blood could serve as a biomarker of exposure to neuropathic OP compounds as well as a predictor of OPIDN and an adjunct to its early diagnosis.     

Comparative effectiveness of organophosphorus protoxicant activating systems in neuroblastoma cells and brain homogenates.

The ability of bromine and rat liver microsomes (RLM) to convert organophosphorus (OP) protoxicants to esterase inhibitors was determined by measuring acetylcholinesterase (AChE) and neuropathy target esterase (NTE) inhibition. Species specific differences in susceptibility to esterase inhibition were determined by comparing the extent of esterase inhibition observed in human neuroblastoma cells and hen, bovine, and rodent brain homogenates. OP protoxicants examined included tri-o-tolyl phosphate (TOTP), O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN), leptophos, fenitrothion, fenthion, and malathion. Bromine activation resulted in greater AChE inhibition than that produced by RLM activation for equivalent concentrations of fenitrothion, malathion, and EPN. For EPN and leptophos, bromine activation resulted in greater inhibition of NTE than RLM. Only preincubation with RLM activated TOTP; resultant inhibition of AChE was less in hen brain (13 +/- 3%) than in neuroblastoma cells (73 +/- 1%) at 10(-6) M. In contrast, 10(-6) M RLM-activated TOTP produced more inhibition of hen brain NTE (89 +/- 6%) than NTE of human neuroblastoma cells (72 +/- 7%). Human neuroblastoma cells and brain homogenates from hens, the accepted animal model for study of OP-induced neurotoxicity, were relatively similar in sensitivity to esterase inhibition. Homogenates from hens were more sensitive to NTE inhibition induced by phenyl saligenin phosphate (PSP), an active congener of TOTP, than were homogenates from less susceptible species (mouse, rat, bovine). AChE of hen brain homogenates was also more sensitive than homogenates from other species to malaoxon, the active form of malathion.     

Constructs of human neuropathy target esterase catalytic domain containing mutations related to motor neuron disease have altered enzymatic properties

Neuropathy target esterase (NTE) is a phospholipase/lysophospholipase associated with organophosphorus (OP) compound-induced delayed neurotoxicity (OPIDN). Distal degeneration of motor axons occurs in both OPIDN and the hereditary spastic paraplegias (HSPs). Recently, mutations within the esterase domain of NTE were identified in patients with a novel type of HSP (SPG39) designated NTE-related motor neuron disease (NTE-MND). Two of these mutations, arginine 890 to histidine (R890H) and methionine 1012 to valine (M1012V), were created in human recombinant NTE catalytic domain (NEST) to measure possible changes in catalytic properties. These mutated enzymes had decreased specific activities for hydrolysis of the artificial substrate, phenyl valerate. In addition, the M1012V mutant exhibited a reduced bimolecular rate constant of inhibition (k_i) for all three inhibitors tested: mipafox, diisopropylphosphorofluoridate, and chlorpyrifos oxon. Finally, while both mutated enzymes inhibited by OP compounds exhibited altered time-dependent loss of their ability to be reactivated by nucleophiles (aging), more pronounced effects were seen with the M1012V mutant. Taken together, the results from specific activity, inhibition, and aging experiments suggest that the mutations found in association with NTE-MND have functional correlates in altered enzymological properties of NTE.     

Potentiation of Organophosphorus-Induced Delayed Neurotoxicity Following Phenyl Saligenin Phosphate Exposures in 2-, 5-, and 8-Week-Old Chickens

Phenylmethylsulfonyl fluoride (PMSF), a nonneuropathic inhibitorof neurotoxk esterase (NTE), is a known potentiator of organophosphorus-induceddelayed neurotoxicity (OPIDN)- The ability of PMSF posttreatment(90 mg/kg, sc, 4 hr after the last PSP injection) to modifydevelopment of delayed neurotoxicity was examined in 2-, 5-,and 8-week-old White Leghorn chickens treated either one, two,or three times (doses separated by 24 hr) with the neuropathicOP compound phenyl saligenin phosphate (PSP, 5 mg/kg, sc). NTEactivity was measured in the cervical spinal cord 4 hr afterthe last PSP treatment. Development of delayed neurotoxicitywas measured over a 16-day postexposure period. All PSP-treatedgroups exhibited >97% NTE inhibition regardless of age ornumber of OP treatments. Two-week-old birds did not developclinical signs of neurotoxicity in response to either singleor repeated OP treatment regimens nor following subsequent treatmentwith PMSF. Five-week-old birds were resistant to the clinicaleffects of a single PSP exposure and were minimally affectedby repeated doses. PMSF posttreatment, however, significantlyamplified the clinical effects of one, two, or three doses ofPSP. A single exposure to PSP induced slight to moderate signsof delayed neurotoxicity in 8-week-old birds with more extensiveneurotoxicity being noted following repeated dosing. As with5-week-old birds, PMSF exacerbated the clinical signs of neurotoxicitywhen given after one, two, or three doses of PSP in 8-week-oldbirds. Axonal degeneration studies supported the clinical findings:PMSF posttreatment did not influence the degree of degenerationin 2-week-old chickens but resulted in more severe degeneration(relative to PSP only exposure) in cervical cords from both5- and 8-week-old birds. The results indicate that PMSF doesnot alter the progression of delayed neurotoxicity in very young(2 weeks of age) chickens but potentiates PSP-induced delayedneurotoxicity in the presence of 0–3% residual NTE activityin older animals. We conclude that posttreatment with neuropathicor nonneuropathic NTE inhibitors, following virtually completeNTE inhibition by either single or repeated doses of a neuropathicagent in sensitive age groups, can modify both the clinicaland morphological indices of delayed neurotoxicity. This studyfurther supports the hypothesis that potentiation of OPIDN occursthrough a mechanism unrelated to NTE.     

Pretreatment with chlorpromazine prevents phospholipid degradation and creatine kinase depletion in isoproterenol-induced myocardial damage in rats

We investigated the effect of chlorpromazine on myocardial phospholipid content and composition and on myocardial creatine kinase (CK) activity in rats with isoproterenol-induced myocardial damage. A single subcutaneous injection of isoproterenol (40 mg/kg) increased heart weight but decreased myocardial phospholipid content and CK activity 24 h after administration. Total phospholipid content was significantly correlated with myocardial CK activity. The decrease of total phospholipid content was accompanied by an increase of the lysophosphatidylcholine (LPC)/phosphatidylcholine (PC) ratio, indicating that conversion from PC to LPC had occurred. Intraperitoneal injection of chlorpromazine (30 mg/kg) prior to isoproterenol injection inhibited the decrease in total phospholipid content and CK activity and the increase in the LPC/PC ratio, but did not affect the increase in heart weight. Pretreatment with propranolol (20 mg/kg) reversed the effects of isoproterenol. On the other hand, dexamethasone (12 mg/kg) and verapamil (50 mg/kg) had no significant effects on the above parameters of myocardial damage. These results suggest that prevention of myocardial phospholipid disruption inhibits enzyme depletion from the myocardium and that chlorpromazine helps to prevent isoproterenol-induced myocardial damage by improving phospholipid metabolism.     

Interindividual variations in enzymes controlling organophosphate toxicity in man.

1 Interindividual variations in an unexposed population have been defined for five enzymes involved in organophosphate (OP) toxicity. The enzymes measured were: red blood cell acetylcholinesterase (AChE), lymphocyte neuropathy target esterase (NTE), serum cholinesterase (ChE), serum paraoxonase and serum arylesterase. 2 AChE and arylesterase were normally distributed in the population whilst the distribution of NTE, ChE and paraoxonase deviated significantly from normal. 3 Assay precision and intra-individual variability were measured for each of the enzymes; the effect on interindividual variation was assessed. 4 Variations in enzyme activities between individuals could have profound effects on susceptibility to OP toxicity. Prior determination of these enzymes may be predictive of susceptibility. 5 Lymphocyte NTE has some limitations as an indicator of exposure to neurotoxic OPs.     

Simultaneous analysis of phospholipid in rabbit bronchoalveolar lavage fluid by liquid chromatography/mass spectrometry

A method was developed to separate and simultaneously quantitate phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC) in rabbit bronchoalveolar lavage fluid (BALF) by liquid chromatography/mass spectrometry (LC/MS). This method consisted of a simple liquid-liquid extraction procedure, separation of phospholipid classes on silica gel column by gradient mode, and detection of mass spectrometry with electrospray ionization (ESI). The precision, accuracy and recovery ranged from 1.6 to 7.6%, -0.8 to +14.7% and 69.3 to 90.0%, respectively. This method was applied to compare the content and the composition of phospholipid classes in BALF collected from inflammation-model and control rabbit. The ratio of LPC concentration to PC significantly increased in inflammation-model BALF compared with control BALF.     

Neuropathy target esterase in mouse whole blood as a biomarker of exposure to neuropathic organophosphorus compounds

The adult hen is the standard animal model for testing organophosphorus (OP) compounds for organophosphorus compound‐induced delayed neurotoxicity (OPIDN). Recently, we developed a mouse model for biochemical assessment of the neuropathic potential of OP compounds based on brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE) inhibition. We carried out the present work to further develop the mouse model by testing the hypothesis that whole blood NTE inhibition could be used as a biochemical marker for exposure to neuropathic OP compounds. Because brain NTE and AChE inhibition are biomarkers of OPIDN and acute cholinergic toxicity, respectively, we compared NTE and AChE 20‐min IC₅₀ values as well as ED₅₀ values 1 h after single intraperitoneal (i.p.) injections of increasing doses of two neuropathic OP compounds that differed in acute toxicity potency. We found good agreement between the brain and blood for in vitro sensitivity of each enzyme as well for the ratios IC₅₀(AChE)/IC₅₀(NTE). Both OP compounds inhibited AChE and NTE in the mouse brain and blood dose‐dependently, and brain and blood inhibitions in vivo were well correlated for each enzyme. For both OP compounds, the ratio ED₅₀(AChE)/ED₅₀(NTE) in blood corresponded to that in the brain despite the somewhat higher sensitivity of blood enzymes. Thus, our results indicate that mouse blood NTE could serve as a biomarker of exposure to neuropathic OP compounds. Moreover, the data suggest that relative inhibition of blood NTE and AChE provide a way to assess the likelihood that OP compound exposure in a susceptible species would produce cholinergic and/or delayed neuropathic effects. Copyright © 2016 John Wiley & Sons, Ltd.     

Organophosphorus-induced delayed neuropathy: A simple and efficient therapeutic strategy

Organophosphorus (OP) used as pesticides and hydraulic fluids can produce acute poisoning known as OP-induced delayed neuropathy (OPIDN), whose effects take long time to recover. Thus a secure therapeutic strategy to prevent the most serious effects of this poisoning would be welcome. In this study, tri-o-cresyl phosphate (TOCP, 500 mg/kg p.o.) was given to hens, followed or not by nimodipine (1 mg/kg i.m.) and calcium gluconate (Ca-glu 5 mg/kg i.v.). Six hours after TOCP intoxication, neuropathy target esterase (NTE) activity inhibition was observed, peaking after 24 h exceeding 80% inhibition. A fall in the plasmatic calcium levels was noted 12 h after TOCP was given and, in the sciatic nerve, Ca²⁺ fell 56.4% 24 h later; at the same time calcium activated neutral protease (CANP) activity increased 308.7%, an effect that lasted 14 days. Any bird that received therapeutic treatment after TOCP intoxication presented significant signs of OPIDN. These results suggest that NTE may be implicated in the regulation of calcium entrance into cells being responsible for the maintenance of normal function of calcium channels, and that increasing CANP activity is responsible to triggering OPIDN. Thus, with one suitably adjusted dose of nimodipine as well as Ca-glu, we believe that this treatment strategy may be used in humans with acute poisoning by neuropathic OP.