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.     

Relative inhibitory potencies of chlorpyrifos oxon,chlorpyrifos methyl oxon,and mipafox for acetylcholinesterase versus neuropathy target esterase

The relative inhibitory potency (RIP) of an organophosphorus (OP) inhibitor against acetylcholinesterase (AChE) versus neuropathy target esterase (NTE) may be defined as the ratio [k(i)(AChE)/k(i)(NTE)], where k(i) is the bimolecular rate constant of inhibition for a given inhibitor against each enzyme. RIPs greater than 1 correlate with the inability of ageable OP inhibitors or their parent compounds to produce OP compound-induced delayed neurotoxicity (OPIDN) at doses below the LD50. The RIP for chlorpyrifos oxon (CPO) is >1 for enzymes from hen brain homogenate, and the parent compound, chlorpyrifos (CPS), cannot produce OPIDN in hens at sublethal doses. This study was carried out to test the hypothesis that the RIP for the methyl homologue of CPO, chlorpyrifos methyl oxon (CPMO), is >1 and greater than the RIP for CPO. Mipafox (MIP), an OP compound known to produce OPIDN, was included for comparison. Hen brain microsomes were used as the enzyme source, and k(i) values (mean +/- SE, microM(-1) min(-1)) were determined for AChE and NTE (n = 3 and 4 separate experiments, respectively). The k(i) values for CPO, CPMO, and MIP against AChE were 17.8 +/- 0.3, 10.9 +/- 0.1, and 0.00429 +/- 0.00001, respectively, and for NTE were 0.0993 +/- 0.0049, 0.0582 +/- 0.0013, and 0.00498 +/- 0.00006, respectively. Corresponding RIPs for CPO, CPMO, and MIP were 179 +/- 9, 187 +/- 4, and 0.861 +/- 0.011, respectively. The results demonstrate that RIPs for CPO and CPMO are comparable, markedly different from that for MIP, and >1, indicating that CPS methyl, like CPS, could not cause OPIDN at sublethal doses.     

Sulfonyl Fluorides and the Promotion of Diisopropyl Fluorophosphate Neuropathy

Phenylmethanesulfonyl fluoride (PMSF) enhances the neuropathicresponse when given to hens after organophosphates causing delayedpolyneuropathy. This study was undertaken to ascertain whetherother sulfonyl fluorides promote diisopropyl fluorophosphate(DFP) neuropathy in hens and if they inhibit neuropathy targetesterase (NTE), the target for organophosphate-induced delayedpolyneuropathy. Among seven sulfonyl fluoride analogs of PMSF(alkyl-, and phenylsulfonyl fluorides), only n-butanesulfonylfluoride was found to be an NTE inhibitor in vitro at a concentration(I₅₀=60 µM) similar to that of PMSF. n-Butanesulfonylfluoride (0.2 mmolkg^–1 sc to hens) caused both NTE inhibitionin nervous tissues (>80%) and promotion of neuropathy afterDFP (0.003 mmolkg^–1 sc) similar to those observed afterthe same molar dose of PMSF. These results confirm that, sofar, all known promoters of organophosphate polyneuropathy arealso NTE inhibitors.     

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.     

Further studies toward a mouse model for biochemical assessment of neuropathic potential of organophosphorus compounds

Inhibition and aging of neuropathy target esterase (NTE) by neuropathic organophosphorus (OP) compounds triggers OP compound‐induced delayed neuropathy (OPIDN), whereas inhibition of acetylcholinesterase (AChE) produces cholinergic toxicity. The neuropathic potential of an OP compound is defined by its relative inhibitory potency toward NTE vs. AChE assessed by enzyme assays following dosing in vivo or after incubations of direct‐acting compounds or active metabolites with enzymes in vitro. The standard animal model of OPIDN is the adult hen, but its large size and high husbandry costs make this species a burdensome model for assessing neuropathic potential. Although the mouse does not readily exhibit clinical signs of OPIDN, it displays axonal lesions and expresses brain AChE and NTE. Therefore, the present research was performed as a further test of the hypothesis that inhibition of mouse brain AChE and NTE could be used to assess neuropathic potential using mouse brain preparations in vitro or employing mouse brain assays following dosing of OP compounds in vivo. Excellent correlations were obtained for inhibition kinetics in vitro of mouse brain enzymes vs. hen brain and human recombinant enzymes. Furthermore, inhibition of mouse brain AChE and NTE after dosing with OP compounds afforded ED₅₀ ratios that agreed with relative inhibitory potencies assessed in vitro. Taken together, results with mouse brain enzymes demonstrated consistent correspondence between in vitro and in vivo predictors of neuropathic potential, thus adding to previous studies supporting the validity of a mouse model for biochemical assessment of the ability of OP compounds to produce OPIDN. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of the Relative Inhibition of Acetylcholinesterase and Neuropathy Target Esterase in Rats and Hens Given Cholinesterase Inhibitors

Inhibition of neuropathy target esterase (NTE, neurotoxic esterase)and acetylcholinesterase (AChE) activities was compared in brainand spinal cords of adult While Leghorn hens and adult maleLong Evan rats 4–48 hr after admiriistration of tri-ortho-tolylphosphate (TOTP po, 50–500 mg/kg to hens; 300–1000mg/kg to rats), phenyl saligenin phosphate (PSP im 0.1–2.5mg/kg to hens; 5–24 mg/kg to rats), mipafox (3–30mg/kg ip to hens and rats), diisopropyl phosphorofluoridate(DFP sc, 0.25–1.0 mg/kg to hens; 1–3 mg/kg to rats),dichlorvos (5–60 mg/kg ip to hens; 5–30 mg/kg torats), malathion (75–300 mg/kg po to hens; 600–2000mg/kg to rats), and carbaryl (300–560 mg/kg ip to hens;30–170 mg/kg to rats). Inhibitions of NTE and AChE weredose-related after administration of all compounds to both species.Hens and rats given TOTP, PSP, mipafox, and DFP demonstrateddelayed neuropathy 3 weeks later, with spinal cord lesions andclinical signs more notable in hens. Ratios of NTE/AChE inhibitionin hen spinal cord, averaged over the doses used, were 2.6 afterTOTP, 5.2 after PSP, 1.3 after mipafox, and 0.9 after DFP, whichcontrast with 0.53 after dichlorvos, 1.0 after malathion, and0.46 after carbaryl. Rat NTE/AChE inhibition ratios were 0.9after TOTP, 2.6 after PSP, 1.0 after mipafox, 0.62 after DFP,1.3 after dichlorvos, 2.2 after malathion, and 1.1 after carbaryl.The lower NTE/AChE ratios in rats given dosages of the fourorganophosphorus compounds that caused delayed neuropathy interferredwith survival, an effect that was not a problem in hens. Thisobservation, along with the absence of overt and specific clinicalsigns and the restricted presence of neuropathological lesionsin rats, suggests that the hen remains the animal of choicefor testing for organophosphorus-induced delayed neuropathy.     

Quantitative structure-activity relationships predict the delayed neurotoxicity potential of a series of O-alkyl-O-methylchloroformimino phenylphosphonates

Inhibition of acetylcholinesterase (AChE) versus inhibition and aging of neuropathy target esterase (NTE) by organophosphorus (OP) compounds in vivo can give rise to distinct neurological consequences: acute cholinergic toxicity versus OP compound-induced delayed neurotoxicity (OPIDN). Previous work has shown that the relative potency of an OP compound to react with NTE versus AChE in vitro may predict its capability to produce OPIDN. The present study was conducted to evaluate further the validity of such predictions and to enhance them with quantitative structure-activity relationships (QSAR) using a homologous series of alkyl phenylphosphonates (RO)C6H5P(O)ON = CCICH3 (PhP; R = alkyl). Neuropathic potential of PhP was assessed by measuring ki(NTE)ki(AChE) ratios in vitro and comparing these with ED50 ratios in vivo. Selectivity for NTE increased with rising R-group hydrophobicity. The ki(NTE)/ki(AChE) ratios were 0.42 (methyl), 3.6 (ethyl), 15 (isopropyl), 36 (propyl), 69 (isobutyl), 105 (butyl), and 124 (pentyl). Ratios > 1 suggest the potential to produce OPIDN at doses lower than the LD50. Inhibition of NTE and AChE in hen brain in vivo was studied 24 h after i.m. injection of hens with increasing doses of methyl and butyl derivatives. Analysis of dose-response curves yielded ED50(AChE)/ED50(NTE) ratio of 0.86 for methyl PhP and 22.1 for butyl PhP. These results predict that the butyl derivative should be more neuropathic than the methyl analogue. Excellent correspondence between in vivo and in vitro predictions of neuropathic potential indicate that valid predictive QSAR models may be based on the in vitro approach. Adoption of this system would result in reducing experimental animal use, lowering costs, accelerating data production, and enabling standardization of a biochemically based risk assessment of the neuropathic potential of OP compounds.     

Effects of Chlorpyrifos on High-Affinity Choline Uptake and [3H]Hemicholinium-3 Binding in Rat Brain

High, subcutaneous doses of the organophosphorus insecticidechlorpyrifos (CPF) in adult male rats can be well-tolerateddespite extensive and persistent acetylcholinesterase (AChE)inhibition. We propose that changes in acetylcholine synthesiscould modulate the toxicity associated with extensive AChE inhibitionfollowing CPF exposure. High-affinity choline uptake (HACU,the rate-limiting step in acetylcholine synthesis) and bindingto [³H]-hemicholinium-3 (HC-3, a specific ligand for the cholinetransporter) were chosen as indicators of acetylcholine synthesis.Female, Sprague-Dawley rats (220–280 g) were treated witheither vehicle (peanut oil, 2 ml/kg, sc) or CPF (280 mg/kg,2 ml/kg, sc), examined daily for clinical signs of toxicity,and sacrificed 1, 2, or 7 days later for neurochemical measurements{AChE inhibition, muscarinic receptor binding using [³H]quinuclidinylbenzilate (QNB) and [³H]cis-methyldioxolane (CD) as ligands,HACU and [³H]HC-3 binding} in frontal cortex. Despite extensiveAChE inhibition (90–93%) at all time points, relativelyminor degrees of overt toxicity were noted in CPF-treated rats.Binding to the non-selective muscarinic antagonist [³H]QNB wasreduced (10–34%), whereas binding to the putative m2-selectiveagonist [³H]CD was increased (15–23%) at all three timepoints. HACU was reduced (20%) in crude synaptosomes preparedfrom CPF-treated rats 1 day following exposure but no significantchanges were noted at 2 or 7 days after treatment. CPF-oxon,the active oxidative metabolite of CPF, was a weak inhibitorof HACU in vitro (IC₅₀>200 µM). Binding to [³H]HC-3was reduced in a dose-related manner 1 day after CPF exposure.Kinetic analyses of [³H]HC-3 binding 1 day after CPF (280 mg/kg)indicated a significant reduction in density {B_max: control,187±18 fmol/mg protein; CPF, 104±12 fmol/mg protein)with no apparent change in binding affinity (K_d: control, 25±3nM; CPF, 19±3 nM). These results suggest that a reductionin HACU/acetylcholine synthesis may contribute, along with compensatorychanges in cholinergic receptors, to the diminished toxicityfollowing extensive AChE inhibition by CPF.     

Comparative in vitro study of the inhibition of human and hen esterases by methamidophos enantiomers

The current Organisation for Economic Co-operation and Development (OECD) guidelines for evaluating organophosphorus-induced delayed neuropathy (OPIDN) require the observation of dosed animals over several days and the sacrifice of 48 hens. Adhering to these protocols in tests with enantiomers is difficult because large quantities of the compound are needed and many animals must be utilized. Thus, developing an in vitro screening protocol to evaluate chiral organophosphorus pesticides (OPs) that can induce delayed neuropathy is important. This work aimed to evaluate, in blood and brain samples from hens, human blood, and human cell culture samples, the potential of the enantiomeric forms of methamidophos to induce acetylcholinesterase (AChE) inhibition and/or delayed neurotoxicity. Calpain activation was also evaluated in the hen brain and SH-SY5Y human neuroblastoma cells. The ratio between the inhibition of neuropathy target esterase (NTE) and AChE activities by the methamidophos enantiomers was evaluated as a possible indicator of the enantiomers' abilities to induce OPIDN. The (-)-methamidophos exhibited an IC(50) value approximately 6 times greater than that of the (+)-methamidophos for the lymphocyte NTE (LNTE) of hens, and (+)-methamidophos exhibited an IC(50) value approximately 7 times larger than that of the (-)-methamidophos for the hen brain AChE. The IC(50) values were 7 times higher for the human erythrocyte AChE and 5 times higher for AChE in the SH-SY5Y human neuroblastoma cells. Considering the esterases inhibition and calpain results, (+)-methamidophos would be expected to have a greater ability to induce OPIDN than the (-)-methamidophos in humans and in hens.     

Toxaphene Inhibition of Calmodulin-Dependent Calcium ATPase Activity in Rat Brain Synaptosomes

Toxaphene Inhibition of Calmodulin-Dependent Calcium ATPaseActivity in Rat Brain Synaptosomes. PRASADA RAO, K. S., TROTTMAN,C. H., MORROW, W., AND DESAIAH, D. (1986) Fundam. Appl. Toxicol.6, 648–653. Effect of toxaphene on Ca⁺²-ATPase activityin rat brain synaptosomes was studied in vitro and in vivo.Ca⁺²-ATPase in calmodulin-depleted synaptosomes was inhibitedin vitro to a maximum of about 50% at 150 µM toxaphenc.Substrate activation kinetics of Ca⁺²-ATPase in synaptosomesrevealed that toxaphene inhibited the enzyme activity noncompetetivelyby decreasing V_max values, without affecting the enzyme-substrateaffinity. Toxaphene inhibited the calmodulin activated Ca⁺²-ATPaseactivity in a concentration-dependent manner with an IC50 of10 µM, a concentration at which no significant effectwas observed on basal enzyme activity. Nuclear and P₂ fraction(synaptosomes) calmodulin levels were reduced significantlyin toxaphene-treated rats. The synaptosomal Ca⁺²-ATPase wasalso reduced to about 45% in toxaphene-treated rats and theactivity was restored to normal levels by the exogenously addedcalmodulin. These results suggest that toxaphene may cause synapticdysfunction by in terfering with calmodulin and its regulationof neuronal calcium.     

An in Vitro Comparison of Rat and Chicken Brain Neurotoxic Esterase

An in Vitro Comparison of Rat and Chicken Brain Neurotoxic Esterase.NOVAK, R., AND PADLLA, S. (1986). Fundam. Appl. Toxicol. 6,464–471. A systematic comparison was undertaken to characterizeneurotoxic esterase (NTE) from rat and chicken brain in termsof inhibitor sensitivities, pH optima, and molecular weights.Paraoxon titration of phenyl valerate (PV)-hydrolyzing carboxylesterasesshowed that rat esterases were more sensitive than chicken toparaoxon inhibition at concentrations <1 µM and superimposablewith chicken esterases at concentrations of 2.5–1000 µM.Mipafox titration of the paraoxon-resistant esterases at a fixedparaoxon concentration of 100 µM (mipafox concentration:0-1000 µM) resulted in a mipafox 150 of 7.3 µM forchicken brain NTE and 11.6 µM for rat brain NTE. NTE (i.e.,paraoxon-resistant, mipafox-sensitive esterase activity) comprised80% of chicken and 60% of rat brain paraoxon-resistant activitywith the specific activity of chicken brain NTE approximatelytwice that of rat brain NTE. The pH maxima for NTE from bothspecies was similar showing broad, slightly alkaline optimafrom pH 7.9 to 8.6. [³H]Diisopropyl phosphorofluoridate (DFP)-labeledNTE from the brains of both species had an apparent mol wt of160,000 measured by sodium dodecyl sulfate polyacrylamide gelelectrophoresis. In conclusion, NTE from both species was verysimilar, with the mipafox 150 for rat NTE within the range ofreported values for chicken and human NTE, and the inhibitorparameters of the chicken NTE assay were applicable for therat NTE assay.     

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.     

Differences in the Toxicity of Soman in Various Strains of Mice

Differences in Toxicity of Soman in Various Strains of Mice.Clement, J.G., Hand, B.T. and Shiloff, J.D. (1981). Fundam.Appl. Toxicol. 1:419–420. The acute toxicity of somanwas assessed in eight strains of mice (ALAS, CD® -1, C57BL,CF1®, CFW® C3H, DBA and BALB/c). In fasted animals theLD₅₀ values for soman varied from 98 µg/kg in C57BL miceto 151 µg/kg in BALB/c mice. In general in non-fastedmice the soman LD⁵⁰ was not significantly changed except inALAS strain where the soman LD⁵⁰ value increased significantly.The different sensitivities to soman poisoning among the variousstrains does not appear to be due totally to differences inlevel of brain acetylcholinesterase. Fasting had no significanteffect on the activity of brain acetylcholinesterase and somantoxicity in CD® -1 mice whereas, upon fasting ALAS strainmice for 18 hr, there was a 25% decrease in brain acetylcholinesterasewhich could explain their increased sensitivity to soman however,it is possible that other biochemical changes may also playa role.     

Biochemical, histopathological and clinical evaluation of delayed effects caused by methamidophos isoforms and TOCP in hens: Ameliorative effects using control of calcium homeostasis

This work evaluated the potential of the isoforms of methamidophos to cause organophosphorus-induced delayed neuropathy (OPIDN) in hens. In addition to inhibition of neuropathy target esterase (NTE) and acetylcholinesterase (AChE), calpain activation, spinal cord lesions and clinical signs were assessed. The isoforms (+)-, (±)- and (-)-methamidophos were administered at 50mg/kg orally; tri-ortho-cresyl phosphate (TOCP) was administered (500mg/kg, po) as positive control for delayed neuropathy. The TOCP hens showed greater than 80% and approximately 20% inhibition of NTE and AChE in hen brain, respectively. Among the isoforms of methamidophos, only the (+)-methamidophos was capable of inhibiting NTE activity (approximately 60%) with statistically significant difference compared to the control group. Calpain activity in brain increased by 40% in TOCP hens compared to the control group when measured 24h after dosing and remained high (18% over control) 21 days after dosing. Hens that received (+)-methamidophos had calpain activity 12% greater than controls. The histopathological findings and clinical signs corroborated the biochemical results that indicated the potential of the (+)-methamidophos to be the isoform responsible for OPIDN induction. Protection against OPIDN was examined using a treatment of 2 doses of nimodipine (1mg/kg, i.m.) and one dose of calcium gluconate (5mg/kg, i.v.). The treatment decreased the effect of OPIDN-inducing TOCP and (+)-methamidophos on calpain activity, spinal cord lesions and clinical signs.     

The Effects of Select Neurotoxic Chemicals on Synaptosomal Monoamine Uptake and K+-Dependent Phosphatase

The Effects of Select Neurotoxic Chemicals on Synaptosomal MonoamineUptake and K⁺-Dependent Phosphatase. Bracken, William M., Sharma,Raghubir P. and Kleinschuster, Stephen J. (1981). Fundam. Appl.Toxicol. 1:432–436. The in vitro inhibition of norepinephrine(NE) and serotonin (5-HT) uptake into rat brain synaptosomesby a diverse group of neurotoxic chemicals was studied. Thetest chemicals included CH₃HgCl, Hg(NO₃)₂, CdCl₂, diisopropylfluorophosphate(DFP), paraoxon, acrylamide and Kepone while chlorpromazineand ouabain were used as reference chemicals. Methylmercuricchloride, Hg(NO₃)₂ and Kepone inhibited the NE and 5-HT uptakewith IC₅₀'s (concentration of chemical inhibiting 50% of uptake)between 10^–4 to 10^–3 M for both amines. Maximalinhibition was 60–100% at 10 3 M. Cadmium chloride, paraoxon,DFP and acrylamide were not inhibitory. The influence of thetest chemicals on synaptosomal K⁺-dependent phosphatase wasstudied. Methylmercuric chloride, Hg(NO₃)₂, CdCl₂ and Keponewere inhibitors of the phosphatase with 50% inhibition (I₅₀)at micromolar concentrations. The phosphatase was most sensitiveto Hg(NO₃)₂ inhibition with an I₅₀ of 0.03 /M. The inhibitoryconcentrations for these chemicals ranged from 10^–7 to10^–3 M. A correlation of the phosphatase and monoamineuptake inhibitions was not suggested from the data. The lowaffinity inhibition (IC₅₀ greater than 10(^–5 M) of theNE and 5-HT uptake by CH₃HgCl, Hg(NO₃)₂ and Kepone suggestedthat this is not a biologically important phenomena. The apparenthigh affinity inhibition (I₅₀ less than 10^–5 M) of thephosphatase demonstrated the specific influences the test compoundscan have on enzymatic processes. Such enzymatic inhibition wouldbe of critical importance if these neurotoxicants were ableto penetrate the synaptic or neuronal membrane.     

Efficacy of Mono- and Bis-Pyridinium Oximes Versus Soman, Sarin and Tabun Poisoning in Mice

Efficacy of Mono- and Bis-Pyridinium Oximes Versus Soman, Sarinand Tabun Poisoning in Mice. Clement, J.G. (1983). Fundam. Appl.Toxicol. 3:533–535. Various oximes (PAM, toxogonin, TMB-4,HS-6, HI-6, HGG-12, HGG-42) combined with atropine were comparedas antidotes of soman, sarin and tabun poisoning in non-fastedCD-1^® male mice. TMB-4 was the most toxic oxime with ani.p. LD₅₀ value of 80 mg/kg and HI-6 was the least toxic oximewith an i.p. LD₅₀ of 588 mg/kg. Upon comparing ED₅₀ values,HGG-42 was the most effective oxime versus soman and tabun poisoningwhereas, HI-6 was the most effective oxime versus sarin poisoning.Further research needs to be done to explain the distinct differencesin efficacy of the oximes versus poisoning by soman, sarin ortabun.     

Subchronic Delayed Neurotoxicity Evaluation of Jet Engine Lubricants Containing Phosphorus Additives

Synthetic polyol-based lubricating oils containing 3% of eithercommercial tricresyl phosphate (TCP), triphenylphosphorothionate(TPPT), or butylated triphenyl phosphate (BTP) additive wereevaluated for neurotoxicity in the adult hen using clinical,biochemical, and neuropathological endpoints. Groups of 17–20hens were administered the oils by oral gavage at a "limit dose"of 1 g/kg, 5 days a week for 13 weeks. A group of positive controlhens was included which received 7.5 mg/kg of one isomer ofTCP (tri-ortho-cresyl phosphate, TOCP) on the same regimen,with an additional oral dose of 500 mg/kg given 12 days beforethe end of the experiment. A negative control group receivedsaline. Neurotoxic esterase (NTE) activity in brain and spinalcord of hens dosed with the lubricating oils was not significantlydifferent from saline controls after 6 weeks of treatment. After13 weeks of dosing, NTE was inhibited 23 to 34% in brains oflubricant-treated hens. Clinical assessments of walking abilitydid not indicate any differences between the negative controlgroup and lubricant-treated hens. Moreover, neuropathologicalexamination revealed no alterations indicative of organophosphorus-induceddelayed neuropathy (OPIDN). in hens treated with the positivecontrol, significant inhibition of NTE was observed in brainand spinal cord at both 6 and 13 weeks of dosing; this groupalso demonstrated clinical impairment and pathological lesionsindicative of OPIDN. In conclusion, the results of the presentstudy indicated that synthetic polyol-based lubricating oilscontaining up to 3% TCP, TPPT, or BTP had low neurotoxic potentialand should not pose a hazard under realistic conditions of exposure.     

Use of the Biventer Cervicis Nerve-Muscle Preparation to Detect Early Changes following Exposure to Organophosphates Inducing Delayed Neuropathy

Use of the Biventer Cervicis Nerve-Muscle Preparation to DetectEarly Changes following Exposure to Organophosphates InducingDelayed Neuropathy. EL-FAWAL, H. A. N., JORTNER, B. S., ANDEHRICH, M.(l990). Fundam. Appl. Toxicol. 15, 108–120.Indices of organophosphorus (OP)-induced delayed neuropathy(OPIDN) in the hen model have traditionally been restrictedto the early inhibition of neuropathy target esterase (NTE)and ataxia with associated pathological changes in hind limbperipheral nerve which occur more than 7 days after OP exposure.The biventer cervicis nerve-muscle preparation was used to evaluateOPIDN in adult hens at various time periods after treatmentwith either the protoxicant tri-o-tolyl phosphate (TOTP), 360mg/kg po, or the active congener phenyl saligenin phosphate(PSP), 2.5 mg/kg im. NTE activity was 21 and 48% of controlfor TOTP and PSP, respectively, 4 days after administration.Clinical signs were notable by 10 days and progressed in severityto paralysis by 21 days. Partial clinical recovery was evidentat 37 days. Denervation hypersensitivity of biventer cervicismuscle to acetylcholine (ACh) was evident as early as 4 daysfollowing TOTP or PSP treatment. The sensitivity to ACh wasgreatest 21 days after OP administration, with partial recoveryat 37 days. Strength-duration curves (SDC) of preparations fromOP-treated hens showed an increase in excitability thresholdsand elevated rheobase with shorter chronaxie than did preparationsfrom controls as early as 4 days following treatment with eithercompound. SDC at 37 days indicated partial reinnervation. Peripheralnerve myelinated fiber degeneration and regeneration consistentwith these physiological changes was seen on histopathologicalexamination. This study suggests that the biventer cervicisnerve-muscle preparation may prove useful for detection of functionaland morphological changes that occur during the interval betweenNTE inhibition and appearance of clinical deficits.     

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.     

Effects of fenthion, fenitrothion and desbromoleptophos on gait, acetylcholinesterase, and neurotoxic esterase in young chicks after in ovo exposure

Previous studies have demonstrated that gait is affected in chicks exposed to organophosphorus esters (OPs) that induce delayed neurotoxicity (OPIDN) in adult hens. To investigate the developmental relationship between such functional deficits and OPIDN, chicks were exposed to 3 OPs with different OPIDN potential. Desbromoleptophos (DBL) induces OPIDN in adult hens; fenthion (FEN) has uncertain OPIDN potential; fenitrothion (FTR) does not induce OPIDN. Chicks were treated by injection into the egg on day 15 of incubation, after the presumed period of OP-induced structural teratogenesis. AChE and neurotoxic esterase (NTE) were assayed during incubation and in parallel with post-hatching evaluations of gait. DBL, 125 mg/kg in ovo, caused paralysis in 70% of chicks after hatching. The gait of surviving chicks was affected for at least 6 weeks and marked by toes curling under. NTE was inhibited until 10 days post-hatching and AChE until hatching. FEN did not inhibit NTE significantly, but AChE was significantly inhibited until hatching. Chicks exposed as embryos to FEN were hyperactive and aggressive. Gait was still affected 6 weeks after treatment with 3 mg/kg FEN. FTR at 125 mg/kg inhibited AChE until day 10 post-hatching, but neither inhibited NTE nor affected gait. The growth of OP-exposed chicks was not significantly decreased, so the decreased length and increased width of the stride could not be ascribed to stunted growth. We conclude that OPs cause irreversible effects on gait that are not related to their defined neurotoxic effects, since altered gait (1) occurs below the age of sensitivity to OPIDN, (2) is seen in the absence of NTE inhibition and (3) does not invariably accompany AChE inhibition.