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.     

Electrophysiologic changes following treatment with organophosphorus-induced delayed neuropathy-producing agents in the adult hen

Although clinical, pathological, and biochemical effects of organophosphorus-induced delayed neuropathy (OPIDN) have been intensively investigated in the adult hen, detailed electrophysiological studies are lacking. Adult white leghorn hens were treated with a single oral dose of either 30 mg/kg tri-2-cresyl phosphate (TOCP), 750 mg/kg TOCP, 4 mg/kg di-n-butyl-2,2-dichlorovinyl phosphate (DBCV), or 30 mg/kg di-n-butyl-2,2-dichlorovinyl phosphinate (DBCV-P). The 750 mg/kg TOCP and DBCV, but not the 30 mg/kg TOCP and DBCV-P, treatments resulted in clinical signs of OPIDN and mild to marked damage of the tibial nerve 21 days after dose. Twenty-four hr lymphocyte neurotoxic esterase (NTE) inhibition was used as an index of brain NTE inhibition for the various organophosphorus compound (OP) treatment. Twenty-four hr lymphocyte NTE inhibition for 30 mg/kg TOCP, 750 mg/kg TOCP, DBCV, and DBCV-P was 54.1, 87.1, 84.8, and 68.3%, respectively. Twenty-one days after dose, the TOCP-treated hens exhibited some abnormalities in conduction velocity and action potential duration in the tibial or sciatic nerves. No abnormalities were observed in action potential parameters of either the DBCV or DBCV-P treatments. Neurotoxic OP (TOCP and DBCV) treatment resulted in decreased refractoriness in the tibial nerve, increased refractoriness in the sciatic nerve, and elevated strength duration threshold for both nerves. These changes were not present in nerves from DBCV-P (a non-neurotoxic NTE inhibitor)-treated hens. These results suggest that refractory period and strength duration abnormalities in peripheral nerve correlate well with the production of OPIDN and are evident without coincident clinical signs or histopathology.     

Delayed neuropathy and acute toxicity studies with pirimiphos-methyl in the hen

This paper describes studies aimed at determining the acute anticholinergic and delayed neurotoxic potential of the organophosphate insecticide pirimiphos-methyl (O-2-diethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate) in the hen. Delayed neuropathy was assessed by biochemical measurement of neuropathy target esterase (NTE) activities in the brain and spinal cord, clinical signs of neuropathy over two 21-day periods and histological assessment of nervous tissue. Acetylcholinesterase (AChE) activity was also determined in the brain and spinal cord. Hens were given a single oral dose of 100 mg kg-1 pirimiphos-methyl, which was followed by a repeated dose after 21 days. Tri-o-cresyl phosphate (TOCP), 500 mg kg-1, was used as a positive control. All pirimiphos-methyl-treated hens received prophylactic doses of N-methylpyridinium-2-aldoxime methanesulphonate (P2S) and atropine sulphate. Hens dosed with pirimiphos-methyl had very low AChE activities (less than 20% of control) in both the brain and spinal cord, 24 and 48 h after dosing. In the TOCP-treated hens, the activities were about 90% of control. NTE activities in the brain and spinal cord of pirimiphos-methyl-treated hens were identical to those in the controls, while they were profoundly inhibited (greater than 80%) in the TOCP-treated hens. All hens dosed with pirimiphos-methyl showed the expected signs of AChE inhibition and, following recovery, usually by Day 5, no clinical signs of delayed neuropathy were seen. The TOCP-treated hens developed clinical signs of neuropathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organophosphate polyneuropathy and neuropathy target esterase: Studies with methamidophos and its resolved optical isomers

 Methamidophos (O, S-dimethyl phospho rothioamidate) causes polyneuropathy in man and hens. However, experiments in the hen show that lower doses of methamidophos either protect from or promote the neuropathy caused by certain organophosphates. The initiation of neuropathy as well as protection from neuropathy are thought to be related to neuropathy target esterase (NTE), whereas promotion is likely to be due to interactions with another unknown target. Methamidophos is a racemate and we report studies with its resolved optical isomers, aimed at elucidating which isomer is responsible for the described effects. The time-course of acetylcholinesterase (AChE) and NTE activity in nervous tissues of hens after inhibition by single doses of either isomer showed that after D-(+) methamidophos (25 mg/kg PO) peak inhibition of both enzymes was achieved within 24 h (80–90%). However, after L-(−) methamidophos (15 mg/kg PO), peak inhibition (80–90%) was obtained within 24 h for AChE, whereas similar NTE inhibition (120 mg/kg PO) was observed only 4 days after dosing. The minimal neuropathic doses of D-(+) and L-(−) methamidophos were 60 and 120 mg/kg PO, respectively, and correlated with >80% NTE inhibition in nervous tissues. OPIDP initiation by either isomer was slightly promoted by phenylmethanesulfonyl fluoride (120 mg/kg SC). D-(+) Methamidophos (25 mg/kg PO) partially protected from dibutyl dichlorovinylphosphate (DBDCVP) neuropathy (up to 0.8 mg/kg SC). This effect correlated with about 70% NTE inhibition. L-(−) Methamidophos (15 or 60 mg/kg PO) did not protect from DBDCVP neuropathy (0.2–0.8 mg/kg SC). D-(+) and L-(−) methamidophos (25 mg/kg PO) promoted DBDCVP neuropathy (0.4 mg/kg SC), and D-(+) methamidophos (24 mg/kg PO) also promoted DFP neuropathy (0.3 mg/kg SC). These effects were unrelated to the degree of NTE inhibition they caused: about 70% by D-(+) methamidophos and extrapolated to about 10–15% by L-(−) methamidophos. We conclude that when racemic methamidophos is given to hens, initiation and protection from OPIDP is due to the interaction of D-(+) methamidophos with NTE. Promotion of OPIDP is due to both isomers as the result of their interaction with unknown site(s). It is possible that the neuropathy due to racemic methamidophos or isomers is a self promoted neuropathy because the promoting doses of both isomers are much lower than the neuropathic ones, and because the neuropathy they initiate is only slightly promoted by phenylmethanesulfonyl fluoride. Received: 5 July 1994/Accepted: 17 October 1994     

Characterization of delayed neurotoxicity in the mouse following chronic oral administration of tri-o-cresyl phosphate

The sensitivity of the mouse to organophosphorus-induced delayed neurotoxicity (OPIDN) has been investigated. One group of five mice received two single 1000-mg/kg po doses of tri-o-cresyl phosphate (TOCP) at a 21-day interval (on Days 1 and 21 of the study); a second group of five mice was given 225 mg/kg of TOCP daily for 270 days. A third group of five animals served as an untreated control. All animals were killed 270 days after the start of the experiment. Daily po dosing of 225 mg/kg TOCP caused a decrease in body weight gain, muscle wasting, weakness, and ataxia which progressed to severe hindlimb paralysis at termination. On the other hand, po administration of two single 1000-mg/kg doses of TOCP at a 21-day interval produced no observable adverse effects. Brain acetylcholinesterase (AChE) and neurotoxic esterase (NTE) activity were 35 and 10% of the control, respectively, in daily dosed animals while AChE and NTE in mice receiving two single 1000-mg/kg doses of TOCP were not significantly altered from the control group. Plasma butyrylcholinesterase activity was 12% of the control group in daily dosed animals. Hepatic microsomal enzyme activities of aniline hydroxylase and p-chloro-N-methylaniline demethylase and NADPH-cytochrome P-450 content in daily dosed animals were increased (141 to 161% of the control group) when compared to controls and mice receiving two single 1000-mg/kg doses of TOCP; the latter being not significantly different from each other. Degeneration of the axon and myelin of the spinal cord and sciatic fascicle were observed and were consistent with OPIDN. This study demonstrates that chronic dosing of TOCP produces OPIDN and induces hepatic microsomal enzyme activity in mice. It is concluded that while the mouse is susceptible to OPIDN, it is a less sensitive and a less appropriate test animal for studying this effect when compared to the adult hen.     

Changes in beclin-1 and micro-calpain expression in tri-ortho-cresyl phosphate-induced delayed neuropathy

Tri-ortho-cresyl phosphate (TOCP) can cause toxic neuropathy known as organophosphate-induced delayed neuropathy (OPIDN), which is pathologically characterized by the swollen axon containing aggregations of neurofilaments, microtubules, and multivesicular vesicles. Autophagy is a self-degradative process which plays a housekeeping role in removing misfolded proteins and damaged organelles. The current study was designed to investigate the possible roles of autophagy in the pathogenesis of OPIDN. Adult hens were treated with a dose of 750mg/kg TOCP by gavage, or injected subcutaneously with 60mg/kg phenylmethanesulfonyl fluoride (PMSF) dissolved in DMSO 24h earlier and subsequently treated with TOCP, then sacrificed on the time-points of 0, 1, 5, 10, and 21 days after dosing of TOCP respectively. The levels of beclin-1 and μ-calpain in tibial nerves and spinal cords were determined by immunoblotting. The results showed that in both tissues TOCP increased the expression of μ-calpain while decreased that of beclin-1. When given before TOCP administration, PMSF pretreatment could protect hens against the delayed neuropathy. In the meantime, pretreatment with PMSF reduced calpain expression below basal and increased beclin-1 expression above basal in tibial nerve, whereas it simply returned calpain and beclin-1 expression to their basal levels in spinal cord. In conclusion, the intoxication of TOCP was associated with a significant change of beclin-1 in hen nervous tissues, which suggested that disruption of autophagy-regulated machinery in neurons might be involved in the pathogenesis of OPIDN.     

Organophosphate induced delayed neuropathy in genetically dissimilar chickens: studies with tri-ortho-cresyl phosphate (TOCP) and trichlorfon

Measurements of plasma cholinesterase (pl.ChE), brain cholinesterase (Br.ChE) and brain Neuropathy Target Esterase (Br.NTE) were made in three different lineages of chickens. All birds received toxicants through gavage in a single oral dose between 08:00 and 09:00 h, after overnight fast. Babcock chickens were treated with 800 mg/kg tri-ortho-cresyl phosphate (TOCP) or 80 mg/kg trichlorfon. The TOCP group had 82% Br.NTE inhibition, when compared to the control group, and no birds displayed symptoms of clinical organophosphate-induced delayed neuropathy (OPIDN). Hy-line w36 lineage chickens were given 1600 mg/kg TOCP and despite this higher dose, Br.NTE inhibition was similar that presented by Babcock chickens. Isabrown chickens were given 1600 mg/kg TOCP or 80 mg/kg trichlorfon. At 36 h all trichlorfon treated birds had from 80 to 90% inhibition of Pl.ChE and Br.ChE, when compared to controls. However, Br.NTE was inhibited less than 20%, and there were no clinical signs of OPIDN. All TOCP treated isabrown chickens had more than 80% Br.NTE inhibition while one of them exhibited just light signs of OPIDN, two chickens became totally paralyzed. This finding suggested that chicken strain was important in the appearance of OPIDN. In addition, 70-80% of NTE inhibition was necessary but was not sufficient to produce OPIDN in chickens, since babcock and hy-line w36 chickens exhibited NTE inhibition in the range of 70-80% without clinical signs of OPIDN.     

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.     

In vitro and in vivo assessment of the effect of impurities and chirality on methamidophos-induced neuropathy target esterase aging.

In vitro and in vivo studies evaluated neuropathy target esterase (NTE) inhibition and aging (i.e., loss of reactivation potential) by analytical and technical grade racemic and resolved L-(-) and D-(+) isomers of methamidophos (O,S-dimethyl phosphoramidothioate). For studies in vitro, microsomal protein from phenobarbital-induced livers was isolated from chick embryos and NTE inhibition assays were performed using chick embryo brain homogenate treated with 1 or 5 mM methamidophos (with and without metabolic enzymes); for studies in vivo, hens received 30 to 35 mg/kg methamidophos injected into the pectoral muscle. NTE aging in hens was assessed 24 h later or after 30 min to 1 h incubation in vitro using solutions of potassium fluoride (KF) reactivator. Technical methamidophos produced significantly higher levels of aged-inhibited NTE than analytical methamidophos or isolated optical isomers. In vivo, technical methamidophos produced 61% total NTE inhibition with 18% aged and 43% unaged NTE; hens receiving analytical grade averaged 6% aged, 52% unaged, and 58% total NTE inhibition. Results for 1 mM analytical methamidophos in vitro were 5% aged, 54% unaged, and 59% total inhibition; for 1 mM technical methamidophos, values averaged 11% aged, 50% unaged, and 60% total NTE inhibition. The degree of NTE aging obtained both in vivo and in vitro for the isolated D-(+) and L-(-) isomers never exceeded that obtained using analytical grade. These data indicate that impurities in methamidophos could contribute to OPIDN potential. The in vitro methodology described could be applied to first tier screening for detection of NTE inhibition and aging, thus reducing the need for whole-animal testing for OPIDN.     

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.     

Triphenyl phosphite neurotoxicity in the hen: inhibition of neurotoxic esterase and of prophylaxis by phenylmethylsulfonyl fluoride

The neuropathic syndrome resulting in the cat and the rat from single or multiple doses of the phosphorous acid ester tiphenyl phosphite (TPP) has been reported to differ from the syndrome caused by numerous phosphoric acid esters, which is known as organophosphorous compound-induced delayed neurotoxicity (OPIDN). Since the hen is used to test compounds for OPIDN, we chose to study the neurotoxicity of single subcutaneous doses of TPP using this animal model. TPP (1000 mg/kg) produced progressive ataxia and paralysis which began to develop 5–10 days after dosing. Similar signs were observed when subcutaneous doses of the OPIDN-causing agents tri-o-cresyl phosphate (TOCP) or diisopropyl phosphorofluoridate (DFP) were administered. The minimum neurotoxic dose of TPP was 500 mg/kg. Prior administration of phenylmethylsulfonyl fluoride (PMSF) prevented the development of a neuropathy induced by DFP, but did not fully protect the hens from TPP or TOCP. PMSF slowed, but did not prevent, the neuropathy caused by TOCP. PMSF reduced the neurotoxicity of 500 mg/kg TPP, but increased the neurotoxicity of 1000 mg/kg TPP. TPP was found to be a very potent inhibitor of neurotoxic esterase (NTE), the putative target site for OPIDN, in vitro, with a k_i of about 2.1×10⁵ M^–1min^–1. Equimolar doses of either TPP (1000 mg/kg) and TOCP (1187 mg/kg) caused over 80% inhibition of neurotoxic esterase (NTE) in brain and sciatic nerve. This high level of NTE inhibition persisted for several weeks. This prolonged inhibition probably accounts for the inability of PMSF to block the neurotoxicity of TOCP. The dose-response curve for NTE inhibition 48 h after dosing indicated that a level of 70% inhibition correlated with the neurotoxicity of TPP.Subneurotoxic doses of TPP and DFP were found to have an additive effect which could be blocked by PMSF. These results indicate that TPP can cause OPIDN in the hen. The synergism between PMSF and the higher dose of TPP suggests the presence of a second neurotoxic effect as well.     

Low non-neuropathic tri-o-cresyl phosphate (TOCP) doses inhibit neuropathy target esterase near the neuropathic threshold in n-hexane pretreated hens

Simultaneous intoxication with hexacarbon solvents and organophosphorus compounds has been considered a possible critical factor in some occupational neuropathies and their interactions proved to cause potentiation effects in hens [1-3]. A high degree of inhibition of neuropathy target esterase (NTE) is needed to develop organophosphorus induced polyneuropathy (OPIDP). In this work, the inhibition of NTE, BuChE and AChE by TOCP on control and n-hexane pretreated (7-15 days, 300 mg/kg per day) hens is studied. Using a single TOCP dose of 200 mg/kg, n-hexane pretreated hens showed synergistic effects, but no significant differences were observed in the inhibition of cholinesterases and NTE in brain or spinal cord. With lower TOCP dose (20 mg/kg) statistically significant differences were observed, which were not drastic but could be important because they involved an increase of inhibition up to critical threshold values (from 40-50% to 60-70% inhibition). However, no clinical effects were observed in these animals. Possible mechanisms of neurotoxic interaction are discussed.     

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.     

Phenylmethanesulfonyl fluoride elicits and intensifies the clinical expression of neuropathic insults

It has been recently reported that phenyl-methanesulfonyl fluoride (PMSF) when given to hens after a neuropathic organophosphate (OP) promotes organophosphate-induced delayed polyneuropathy (OPIDP). Chicks are resistant to OPIDP despite high inhibition/aging of neuropathy target esterase (NTE), the putative target of OPIDP initiation. However, when PMSF (300 mg/kg s.c.) is given to chicks after di-butyl 2,2-dichlorovinyl phosphate (DBDCVP, 1 or 5 mg/kg s.c.), OPIDP is promoted. Inhibition/aging of at least 30% of NTE was thought to be an essential prerequisite for promotion to be elicited in adult hens. However, we observed in hens that when NTE is maximally affected (>90%) by phenyl N-methyl N-benzyl carbamate (40 mg/kg i.V.), a non-ageable inhibitor of NTE, and then PMSF is given (120 mg/kg/day s.c. × 3 days) clinical signs of neuropathy become evident. Methamidophos (50 mg/kg p. o. to hens), which produces in vivo a reactivatable form of inhibited NTE, was shown either to protect from or promote OPIDP caused by DBDCVP (0.45 mg/kg s. c), depending on the sequence of dosing. Because very high doses of methamidophos cause OPIDP, we considered this effect to be a “self-promoted” OPIDP. We concluded that NTE inhibitors might have different intrinsic activities for producing OPIDP once NTE is affected. Aging might differentiate highly neuropathic OPs, like DBDCVP, from less neuropathic OPs, like methamidophos, or from the least neuropathic carbamates, which require promotion in order for neuropathy to be expressed. Retrograde axonal transport in motor fibers was measured as the accumulation of¹²⁵ I-tetanus toxin in spinal cord after injection in the gastrocnemius muscle of chicks treated either with DBDCVP (5 mg/kg s.c.) or with DBDCVP followed by PMSF (300 mg/kg s.c). Retrograde axonal transport was reduced in both groups (to about 50%, 10 days after dosing) and returned to normal 27 days after dosing. However, DBDCVP-treated chicks had a mild neuropathy which recovered relatively quickly, whereas chicks to which PMSF was also given had more severe signs which did not recover by day 27. We concluded that promotion affects a site other than NTE and that it acts at a point downstream from initiation. PMSF was also shown to promote 2,5-hexanedione (2,5-HD) neuropathy. 2,5-HD was given to hens at doses (200 mg/kg/day i.p. × 8 days) which caused mild and reversible neuropathy. When PMSF (120 mg/kg/day × 2 days at the end of 2,5-HD treatment) was given, more severe and irreversible signs of neuropathy were observed. We conclude that promotion might be a common feature in neuropathies of different origin. Part of this work was presented at the 30th Annual Meeting of the Society of Toxicology held in Dallas, TX, USA, February – March 1991     

The effect of Calcicol as calcium tonic on delayed neurotoxicity induced by organophosphorus compounds

To examine whether delayed neuropathy is prevented or alleviated when Ca is administered to experimental animals before or after organophosphorus compounds (OPs) dosing, we observed the effects of Calcicol administration as a calcium tonic on delayed neurotoxicity by OPs in hens. The hens (n=28) were randomly divided into seven groups (four in each group). One group received glycerol formal as vehicle group, two groups received 30 mg/kg leptophos or 40 mg/kg triortho-cresyl phosphate (TOCP) (L group and T group), two groups received 2.4 mg/kg Ca(2+) (0.3 ml/kg Calcicol) 24 h before leptophos or TOCP administration, and the last two groups received 2.4 mg/kg Ca after leptophos or TOCP administration, respectively. Although delayed polyneuropathy induced by OPs could not be prevented completely by Calcicol, the clinical signs of organophosphorus-induced delayed neuropathy (OPIDN) in hens that received Calcicol soon before or after OPs administration were less severe than those in hens that received only OPs and there were significant differences in OPIDN score between groups (P<0.05). This shows that polyneuropathy and the recovery function of nerves and muscles suffering from polyneuropathy can be alleviated, as long as calcium tonic is administered before the clinical signs develop. This study offers hope of recovery to humans who are exposed to these OPs because of work, attempted suicide, accidental ingestion or other accidents, etc. Meanwhile, our results indicate further that there is a relationship between a decrease in Ca(2+) concentration in tissues and induction of delayed neuropathy.     

Effect of beta-naphthoflavone on o-tolyl saligenin phosphate-induced delayed neuropathy in two lines of chickens

The effect of the microsomal enzyme inducer beta-naphthoflavone (beta NF) on the development of organophosphorus-induced delayed neuropathy (OPIDN) was examined in two laboratories (VPI and MSU), utilizing two strains of White Leghorn hens. A single intraperitoneal injection of beta NF at 80 mg/kg body weight 48 h prior to administration of o-tolyl saligenin phosphate (TSP), the neuroactive metabolite of tri-o-tolyl phosphate (TOTP), caused a significant increase in hepatic microsomal cytochrome P-450 concentrations and aniline hydroxylase activities after 72 h in both strains. Hepatic carboxylesterase and cholinesterase activities were not affected by beta NF treatment in either strain. Administration of TSP in single subcutaneous doses of 20 and 25 mg/kg body weight (VPI) or 30 and 60 mg/kg body weight (MSU) caused significant inhibition of whole-brain neuropathy target esterase (NTE) activity 24 h postdosing, and hens subsequently developed clinical signs characteristics of OPIDN. beta NF had no significant effect on NTE inhibition or on initiation or severity of OPIDN clinical signs. However, OPIDN clinical signs were less severe in the strain of bird (MSU) with the higher intrinsic hepatic carboxylesterase activity and the higher beta NF-induced cytochrome P-450 concentration. The study indicates that microsomal enzyme induction, which has been shown to alleviate TOTP-induced delayed neuropathy, could not alleviate OPIDN resulting from exposure to TSP. This study also suggests that strain may affect susceptibility to TSP-induced delayed neuropathy.     

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.     

Repeated low doses of O-(2-chloro-2,3,3 trifluorocyclobutyl) O-ethyl S-propyl phosphorothioate (KBR-2822) do not cause neuropathy in hens

Certain esterase inhibitors such as O-(2-chlo-ro-2,3,3-trifluorocyclobutyl) O-ethyl S-propyl phosphorothioate (KBR-2822) and phenylmethanesulfonyl fluoride (PMSF) cause exacerbation (promotion) of toxic and traumatic axonopathies. Although these chemicals are capable of inhibiting neuropathy target esterase (NTE), which is the target for organophosphate induced delayed neuropathy, the target for promotion is unlikely to be NTE. Experiments were aimed to ascertain if neuropathy is caused by repeated dosing with a promoter not causing NTE inhibition and in the absence of deliberate injury to axons. Hens were treated with KBR-2822 (0.2 or 0.4 mg/kg per day) by gavage for 90 days and observed for clinical signs up to 21–23 days after treatment when histopathological examination was carried out. NTE and acetylcholinesterase (AChE) were measured at intervals and mean percentages of inhibition at steady state of inhibition/resynthesis (on day 20) were as follows: mean inhibition NTE was ≤8% in the 0.2 mg/kg group and between 15 and 18% in the 0.4 mg/kg group in brain, spinal cord and peripheral nerve; mean AChE inhibition in brain was 31 and 57% in the two experimental groups, respectively. Controls treated with paraoxon (not neuropathic or a promoter and given at 0.05 mg/kg per day by gavage) showed 45% mean AChE inhibition and no NTE inhibition. Neither clinical nor morphological signs of neuropathy were observed in any group. To ascertain whether sub-clinical lesions were produced by the repeated treatment with KBR-2822, hens were given KBR-2822 (0.2 mg/kg per day) for 21 days by gavage followed by PMSF (120 mg/kg s.c. 24 h after the last dose of KBR-2822). A control group of hens was treated with the neuropathic DFP (0.03 mg/kg s.c. daily for 21 days causing 40–50% NTE inhibition) followed by PMSF (120 mg/kg s.c.). After PMSF, the KBR-2822 treated hens did not develop neuropathy whereas DFP treated hens did. Lack of neuropathy after repeated treatment with KBR-2822 indicates that a continuous promoting `pressure' on hen axons is harmless in the absence of a concurrent biochemical or neurotoxic injury. Received: 22 May 1997 / Accepted: 16 September 1997     

Inhibition of Hen Brain Acetylcholinesterase and Neurotoxic Esterase by Chlorpyrifos in Vivo and Kinetics of Inhibition by Chlorpyrifos Oxon in Vitro: Application to Assessment of Neuropathic Risk

Chlorpyrifos (CPS; O,O-diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate;Dursban) is a widely used broad-spectrum organophosphorus (OP)insecticide. Because some OP compounds can cause a sensory-motordistal axonopathy called OP compound-induced delayed neurotoxicity(OPIDN), CPS has been evaluated for this paralytic effect. Earlystudies of the neurotoxicity of CPS in young and adult hensreported reversible leg weakness but failed to detect OPIDN.More recently, a human case of mild OPIDN was reported to resultfrom ingestion of a massive dose (about 300 mg/kg) in a suicideattempt. Subsequent experiments in adult hens (the currentlyaccepted animal model of choice for studies of OPIDN) showedthat doses of CPS in excess of the LD₅₀ in atropine-treatedanimals inhibited brain neurotoxic esterase (NTE) and producedmild to moderate ataxia. Considering the extensive use of CPSand its demonstrated potential for causing OPIDN at supralethaldoses, additional data are needed to enable quantitative estimatesto be made of the neuropathic risk of this compound. Previouswork has shown that the ability of OP insecticides to causeacute cholinergic toxicity versus OPIDN can be predicted fromtheir relative tendency to inhibit the intended target, acetylcholinesterase(AChE), versus the putative neuropathic target, NTE, in braintissue. The present study was designed to clarify the magnitudeof neuropathic risk associated with CPS exposures by measuringhen brain AChE and NTE inhibition following dosing in vivo anddetermining the bimolecular rate constant of inhibition (k₁)for each enzyme by the active metabolite, CPS oxon (CPO), invitro. CPS administered to atropine-treated adult hens at 0,75, 150, and 300 mg/kg po in corn oil produced mean values forbrain AChE inhibition 4 days after dosing of 0, 58, 75, and86%, respectively, and mean values for brain NTE inhibitionof 0, 21, 40, and 77%, respectively. Only the high dose (sixtimes the unprotected LD₅₀ in hens) produced NTE inhibitionabove the presumed threshold of 70%, and these animals werein extremis from cholinergic toxicity at the time of euthanizationdespite continual treatment with atropine. When 150 mg/kg CPSpo in corn oil was given to atropine-treated hens on Day 0,inhibition on Days 1, 2,4, 8, and 16 for brain AChE was 86,82, 72, 44, and 29%, respectively, and for brain NTE was 30,28, 38, 29, and 6%, respectively. No signs of OPIDN were observedin any of the animals during the 16-day study period. Kineticstudies of the inhibition of hen brain AChE and NTE by CPO invitro demonstrated that CPO exhibits high potency and extraordinaryselectivity for its intended target, AChE. The k₁, values were15.5 µM^–1 min^–1 for AChE and 0.145 µM^–1min^–1 for NTE. The calculated fixed-time (20-min) I₅₀values were 2.24 nM for AChE and 239 nM for NTE, yielding anI₅₀ ratio for NTE/AChE of 107. These results may be comparedwith data compiled for other OP compounds with respect to NTE/AChEI₅₀ ratios and the corresponding doses required to produce OPIDNrelative to the LD₅₀. In general, NTE/AChE I₅₀ ratios greaterthan 1 indicate that the dose required to produce OPIDN is greaterthan the LD₅₀. Taken together, the results of this study indicatethat acute exposures to CPS would not be expected to cause OPIDNexcept under extreme conditions such as attempted suicides involvingmedically assisted survival of doses considerably in excessof the LD₅₀.     

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.