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     

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.     

Central-peripheral delayed neuropathy caused by diisopropyl phosphorofluoridate (DFP): segregation of peripheral nerve and spinal cord effects using biochemical, clinical, and morphological criteria

Systemic injection of diisopropyl phosphorofluoridate (DFP; 1 mg/kg, sc) causes delayed neuropathy in hens. This effect is associated with a high level of organophosphorylation of neuropathy target esterase (NTE) followed by an intramolecular rearrangement called "aging." Phenylmethanesulfonyl fluoride (PMSF) also attacks the active center of NTE but "aging" cannot occur. This compound does not cause neuropathy and protects against a subsequent challenge systemic dose of DFP. Intraarterial injection of DFP (0.185 mg/kg) into only one leg of hens caused a high NTE inhibition (greater than 80%) in the sciatic nerve of the injected leg, but not in other parts of the nervous system (37% average). A unilateral neuropathy with typical histopathological lesions developed in the injected leg. PMSF (0.55 mg/kg) injected into each sciatic artery caused 47% inhibition of sciatic nerve NTE but only 17-22% inhibition of NTE elsewhere; it did not produce clinical or histopathological lesions. When these hens were challenged with DFP (1 mg/kg, sc), high inhibition of residual-free NTE (greater than 85%) occurred throughout the nervous system and clinical signs of a syndrome different from the classical delayed neuropathy developed: this spinal cord type of ataxia was associated with histopathological lesions in the spinal cord but not in peripheral nerve. PMSF (1 mg/kg) injected into only one sciatic artery caused selective protective inhibition of sciatic nerve NTE of that leg. After systemic challenge by DFP, clinical effects expressed were a combination of spinal cord ataxia plus unilateral peripheral neuropathy. The challenge dose of DFP (1 mg/kg, sc) was insufficient to produce clear histopathological lesions in unprotected peripheral nerves although spinal lesions were found in these hens. Thus clinical evaluation of the peripheral nervous system by means of walking tests and a simple test of "leg retraction" reflexes was more sensitive and specific in diagnosis of peripheral neuropathy than was the histopathology.     

Promotion of organophosphate-induced delayed polyneuropathy by phenylmethanesulfonyl fluoride

Certain sulfonates, like phenylmethanesulfonyl fluoride (PMSF), carbamates, and phosphinates, when given prior to neuropathic doses of organophosphates such as diisopropyl phosphorofluoridate (DFP), protect hens from organophosphate-induced delayed polyneuropathy (OPIDP). Protection was related to inhibition of the putative target of OPIDP, which is called Neuropathy Target Esterase (NTE). NTE inhibition above 70-80% in the nervous system of hens followed by a molecular rearrangement called aging initiates OPIDP. PMSF and other protective chemicals inhibit NTE but OPIDP does not develop because aging cannot occur. DFP (1 mg/kg sc) inhibited NTE above 70-80% in peripheral nerve and caused OPIDP in hens. Lower doses (0.3 and 0.5 mg/kg sc) caused about 40-60% NTE inhibition and no or marginal OPIDP. Chlorpyrifos (90 mg/kg po) also caused OPIDP. When repeated (30 mg/kg sc daily for 9 days) or single (5-120 mg/kg sc) doses of PMSF were given after either DFP or chlorpyrifos, OPIDP developed in birds treated with nonneuropathic doses of DFP and was more severe in birds treated with chlorpyrifos or higher doses of DFP. PMSF increased NTE inhibition to greater than 90%. Promotion of OPIDP with a single dose of PMSF (120 mg/kg sc) was obtained in birds up to 11 days after a marginally neuropathic dose of DFP (0.5 mg/kg sc). Promotion was also obtained with phenyl N-methyl N-benzyl carbamate (40 mg/kg iv) but not with non-NTE inhibitors in vivo such as paraoxon or benzenesulfonyl fluoride when given at maximum tolerated doses. These results indicate that protection from OPIDP is only one effect of PMSF because promotion of OPIDP is also observed depending upon the sequence of dosing. Either effect is always related to the doses of PMSF, which inhibit NTE.     

Intra-arterial injection of diisopropylfluorophosphate or phenylmethanesulphonyl fluoride produces unilateral neuropathy or protection, respectively, in hens

Hens injected in one sciatic artery with diisopropylfluorophosphate (DFP) (0.184 mg/kg) developed monolateral ataxia on the injected side 10-12 days later. The inhibition of neuropathy target esterase (NTE) was 85% in the sciatic nerve of the injected leg and less than 60% in the contralateral sciatic nerve, in spinal cord and in brain. Other hens injected in the wing vein with the same dose of DFP showed low inhibition of NTE in the nervous system and did not develop delayed neuropathy. Hens injected in one sciatic artery with phenylmethanesulphonyl fluoride (PMSF) (1 mg/kg) and 24 hr later with high subcutaneous dose of DFP (1.1 mg/kg) developed monolateral ataxia 10-12 days later on the side not injected with PMSF. The level of NTE inhibition after PMSF was greater than 40% in the sciatic nerve on the injected side compared with less than 20% in other parts of the nervous system. The same dose of PMSF injected in the wing vein produced low NTE inhibition in the nervous system and failed to protect the animals from the same high systemic dose of DFP. We conclude that both toxic and protective effects of NTE inhibitors for delayed neuropathy are better related to the level of NTE inhibition in the peripheral nerve on the site of injection than to NTE inhibition in other parts of the nervous system. Furthermore we suggest that NTE inhibition should also be measured in the peripheral nerve in the standard toxicity testing for organophosphate-induced delayed neurotoxicity.     

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     

A Microassay Method for Neurotoxic Esterase Determinations

A microtiter plate reader with an associated computer to averagetriplicate samples and subtract blanks was used for readingand calculating neurotoxic esterase (NTE, also known as neuropathytarget esterase) activities in spinal cord regions of hens 4hr after administration of diisopropylphosphorofluoridate (DFP,0.5 mg/kg sc). Although NTE inhibition is an early indicatorof organophosphorus ester-induced delayed neuropathy, DFP-inducedinhibition was not greater in regions of the spinal cord wherepathological changes are most notable. Acetylcholinesterase(AChE) activities and protein determinations were also doneon these tissues using microassay methods. DFP-induced AChEinhibition was similar to NTE inhibition. In addition to thecapability to be used for small regional esterase activity measurements,the microassay was advantageous because the number of samplesincorporated into a single assay was increased and the timeneeded for the NTE assay was reduced by 50%. Total volume ofincubate in each well was 0.3 ml; the incubate contained 1/20quantities of sample and reagents necessary in more conventionalassays. Validation of the microassay was performed by comparisonwith more conventional assays when measuring inhibition of NTEand AChE in brains of control and experimental hens of two differentgenetic strains (B13B13 and B21B21 white leghorns). Experimentalbirds were given DFP, 0.5 mg/kg sc, 24 hr before samples werecollected. NTE activities in brains of control hens were similarusing both types of NTE analytical procedures. Percentage inhibitionof NTE caused by DFP was within 4% using both assay proceduresin both strains of hens. The microassay was sensitive enoughto detect NTE activity in 42 µg of hen brain after 15min of incubation. Hen lymphocytes could also be examined foreffects of organophosphorus esters on NTE activity, with 14.1±2.2 and 8.3 ±2.2 /xmol/15 min/mg protein in 1x10⁶cells measured in samples taken before and 4 hr after administrationof 0.5 mg/kg SC DFP.     

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.     

Selective promotion by phenylmethanesulfonyl fluoride of peripheral and spinal cord neuropathies initiated by diisopropyl phosphorofluoridate in the hen

This paper reports studies in hens showing that diisopropyl phosphorofluoridate (DFP) neuropathy is promoted by PMSF when initiated either in central (spinal cord) or peripheral nervous system. Moreover, the critical site for promotion is in peripheral nerve axons rather than in their cell bodies. Selective promotion in peripheral nerves was achieved by giving PMSF into sciatic artery monolaterally (7 mg/kg) to birds where neuropathy was initiated by DFP, either systemically (0.3 mg/kg s.c.) or intra-arterially (0.04 mg/kg in the same artery). Birds developed monolateral neuropathy in the leg where PMSF was delivered. Promotion of spinal cord neuropathy was achieved by giving PMSF (120 mg/kg s.c.) to birds where neuropathy was initiated selectively in spinal cord. This was obtained by protecting peripheral axons with intra-arterial bilateral injections of PMSF (0.55 × 2 mg/kg) followed by DFP (0.3, 0.4 or 0.7 mg/kg s.c.). The resulting syndrome was characterized by spastic ataxia.     

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.     

Triphenylphosphite neuropathy in hens

Single doses of triphenyl phosphite (TPP), a triester of trivalent phosphorus, cause ataxia and paralysis in hens. Characteristics of neurotoxicity were described as somewhat different from organophosphate induced delayed polyneuropathy (OPIDP), which is caused by triesters of pentavalent phosphorus. The onset of TPP neuropathy was reported to occur earlier than that of OPIDP (5–10 versus 7–14 days after dosing, respectively), and chromatolysis, neuronal necrosis and lesions in certain areas of the brain were found in TPP neuropathy only. Pretreatment with phenylmethanesulfonyl fluoride (PMSF) protects from OPIDP, but it either partially protected from effects of low doses or exacerbated those of higher doses of TPP. In order to account for these differences with OPIDP, it was suggested that TPP neuropathy results from the combination of two independent mechanisms of toxicity: typical OPIDP due to inhibition of neuropathy target esterase (NTE) plus a second neurotoxicity related with other target(s). We explored TPP neuropathy in the hen with attention to the phenomena of promotion and protection which are both caused by PMSF when given in combination with typical neuropathic OPs. When PMSF is given before neuropathic OPs it protects from OPIDP; when given afterwards it exaggerates OPIDP. The former effect is due to interactions with NTE, the latter to interactions with an unknown site. The time course of NTE reappearance after TPP (60 or 90 mg/kg i.v.) inhibition showed a longer half-life when compared to that after PMSF (30 mg/kg s.c.) (10–15 versus 4–6 days, respectively). The clinical signs of TPP neuropathy (60 or 90 mg/kg i.v.) were similar to those observed in OPIDP, appeared 7–12 days after treatment, correlated with more than 70% NTE inhibition/aging and were preceded by a reduction of retrograde axonal transport in sciatic nerve of hens. TPP (60 mg/kg i.v.) neuropathy was promoted by PMSF (120 mg/kg s.c.) given up to 12 days afterwards and was partially protected by PMSF (10–120 mg/kg s.c.) when given 24 h before TPP (60 or 90 mg/kg i.v.). The previously reported early onset of TPP neuropathy might be related to the higher dose used in those experiments and to the resulting more severe neuropathy. The lack of full protection might be explained by the slow kinetics of TPP, which would cause substantial NTE inhibition when PMSF effects on NTE had subsided. Since PMSF also affects the promotion site when given before initiation of neuropathy, the resulting neuropathy would then be due to both protection from and promotion of TPP effects by PMSF. No promotion by PMSF (120 mg/kg s.c.) was observed in TPP neuropathy (90 mg/kg i.v.) partially protected by PMSF (10–30 mg/kg s.c.) This might also be explained by the concurrent effects on NTE and on the promotion site obtained with PMSF pretreatment. We conclude that TPP neuropathy in the hen is likely to be the same as typical OPIDP. The unusual effects of combined treatment to hens with TPP and PMSF are explained by the prolonged pharmacokinetics of TPP and by the dual effect of PMSF i.e. protection from and promotion of OPIDP.     

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.     

Interaction of phosphamidon with neuropathy target esterase and acetylcholinesterase of hen brain

Phosphamidon (PSM) is an organophosphorus insecticide widely used in agriculture. This study was undertaken to examine the interaction of PSM with acetylcholinesterase (AChE) and neuropathy target esterase (NTE) of hen brain in vitro and in vivo. PSM was a potent inhibitor of AChE, with an I₅₀ of 2.9μM and second-order rate constant (k_a) of 1.2×10⁴ M^?1 min^?1 at 37°C. PSM-inhibited AChE aged rapidly (t_1/2=1.9h). Pyridinium oximes pralidoxime, trimedoxime, obidoxime and HI-6 were effective reactivators of PSM-inhibited AChE, providing up to 75% reactivation. PSM was one of the weakest inhibitors of NTE among organophosphorus compounds, with an I₅₀ of 19 mM and k_a of 1.8 M^?1 min^?1 at 37°C. Inhibited NTE did not reactivate spontaneously and KF-induced reactivation was not obtained even at the earliest tested moments, so it was not clear whether aging of PSM-inhibited NTE occurred very quickly or the KF molecule could not affect the stability of phosphoryl-NTE bond. From the ratio of k_as for NTE and AChE (0.00015) it was predicted that delayed neuropathic effects of PSM in vivo would appear only at doses far above the acute LD₅₀. The LD₅₀ value of PSM p.o. for hens was 9 mg/kg. Hens were treated with a single oral dose of PSM, combined with standard antidotal treatment which included atropine, physostigmine, pralidoxime and anticonvulsant midazolam. Doses of 90 and 250 mg/kg caused up to 27% and 45% NTE inhibition 48h after poisoning, respectively. Clinical signs of neuropathy were not seen up to 25 days after treatment, which could be expected, since the proposed level (>70%) of NTE inhibition necessary for the occurrence of delayed neuropathy was not achieved. The results suggest that PSM, at doses tested, has no ability to cause delayed neuropathy in hens without showing signs of severe cholinergic intoxication.     

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.     

Distribution of neurotoxic esterase activity in the brain of control and diisopropyl phosphorofluoridate-treated hens: in vivo and in vitro exposure

Neurotoxic esterase (NTE) is a protein which is hypothesized to be the site where certain organophosphorus compounds act to produce delayed-onset neurotoxicity. Adult white Leghorn hens (Gallus domesticus) were injected subcutaneously (0.5 mg/kg and 2.0 mg/kg) with diisopropyl phosphorofluoridate (DFP). Control and DFP-treated hens were killed 24 h after treatment and their brains sectioned into telencephalic, cerebellar, diencephalic, mesencephalic, metencephalic tegmentum, and myelencephalic portions. NTE activity was highest in the telencephalon and cerebellum, and brainstem activity progressively decreased moving caudally with the myelencephalon approaching reported spinal cord levels. Percent inhibition of NTE by DFP (0.5 mg/kg and 2.0 mg/kg) did not differ among brain regions or whole brain. The IC50's for DFP were not significantly different either among brain regions or whole brain. The results suggest that nervous system regions with higher NTE levels are protected from delayed neuropathy by virtue of overabundant NTE activity.     

In vivo and in vitro regional differential sensitivity of neuropathy target esterase to Di-n-butyl-2,2-dichlorovinyl phosphate

Organophosphate-induced delayed polyneuropathy (OPIDP) is initiated by inhibition/aging of more than 70–75% of neuropathy target esterase (NTE). Di-n-butyl-2,2-dichlorovinyl phosphate (DBDCVP) (1 mg/kg s.c.) inhibited 96%, 86% and 83% of NTE in brain, spinal cord and peripheral nerve, respectively, and induced a typical central peripheral distal axonopathy in hens. A lower dose (0.45 mg/kg s.c.) caused 90%, 83% and 54% NTE inhibition in the same organs; by contrast, hens developed a spastic ataxia with axonal degeneration in spinal cord but not in peripheral nerve. With a dose of 0.2 mg/kg s.c., a suprathreshold inhibition of NTE was produced in brain (78%) but not in spinal cord (56%) and peripheral nerve (33%) and no morphological or clinical signs of neuropathy developed in hens. With doses up to 4.0 mg/kg s.c., acetylcholinesterase (AChE) inhibition was similar throughout the nervous system. In vitro time-course inhibition studies showed a different sensitivity to DBDCVP of NTE from peripheral nerve (k_a = 5.4 × 10⁶) relative to that from spinal cord (k_a = 13.9 × 10⁶) or brain (k_a = 20.6 × 10⁶). In vitro I₅₀s of DBDCVP for AChE were similar in brain, spinal cord and peripheral nerve (11–17 nM). These data support the hypothesis that the critical target for initiation of OPIDP is located in the nerve fiber, possibly in the axon and also suggest that peripheral nerve NTE has a different sensitivity to DBDCVP than the brain enzyme. Moreover, they confirm data showing that the degree of NTE inhibition in brain after dosing with organophosphates may not be a good monitor for the enzyme in parts of the nervous system where axonal degeneration actually develops. Therefore, direct assay of peripheral nerve NTE yields data which closely correlate with degree of axonal degeneration.Part of this work was presented at the 26th Annual Meeting of the Society of Toxicology, held in Washington DC, USA, February 24–27, 1987 and at the International Meeting on Esterases, Hydrolysing Organophosphorus Compounds, held in Dubrovnik, Yugoslavia, April 24–27, 1988     

Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride

It is well known that pretreatment with the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF) can protect experimental animals from organophosphorus-induced delayed neurotoxicity (OPIDN), presumably by blocking the active site of neurotoxic esterase (NTE) such that binding and "aging" of the neuropathic OP is thwarted. We report here that while PMSF (60 mg/kg, sc) given 4 h before the neuropathic organophosphate (OP) mipafox (50 mg/kg, im) completely prevented the clinical expression of OPIDN in hens, the identical PMSF treatment markedly amplified the delayed neurotoxicity (relative to hens treated with OP only) if administered 4 h after mipafox (5 or 50 mg/kg, im). Moreover, in a separate experiment using diisopropylphosphorofluoridate (DFP) as the neurotoxicant in place of mipafox, posttreatment with PMSF 4 h after DFP (0.5 mg/kg) also accentuated the severity of ataxia. These data indicate that PMSF only protects against OPIDN if given prior to exposure to the neurotoxicant; treatment with PMSF after OP exposure critically exacerbates the delayed neurotoxicity from exposure to organophosphorus compounds.     

Effects of S-ethyl hexahydro-1H-azepine-1-carbothioate (molinate) on di-n-butyl dichlorovinyl phosphate (DBDCVP) neuropathy.

Certain esterase inhibitors protect from organophosphate-induced delayed polyneuropathy (OPIDP) when given before a neuropathic organophosphate by inhibiting neuropathy target esterase (NTE). In contrast, they can exaggerate OPIDP when given afterwards and this effect (promotion) is associated with inhibition of another esterase (M200). In vitro sensitivities of hen, rat, and human NTE and M200 to the active metabolites of molinate, sulfone, and sulfoxide, were similar. NTE and M200 were irreversibly inhibited (> 78%) in brain and peripheral nerve of hens and rats given molinate (100-180 mg/kg, sc). No clinical or morphological signs of neuropathy developed in these animals. Hens and rats were protected from di-n-butyl dichlorovinyl phosphate neuropathy (DBDCVP, 1 and 5 mg/kg, sc, respectively) by molinate (180 or 100 mg/kg, sc, 24 h earlier, respectively) whereas 45 mg/kg, sc molinate causing about 34% NTE inhibition offered partial protection to hens. Hens treated with DBDCVP (0.4 mg/kg, sc) developed a mild OPIDP; molinate (180 mg/kg, 24 h later) increased the severity of clinical effects and of histopathology in spinal cord and in peripheral nerves. Lower doses of molinate (45 mg/kg, sc), causing about 47% M200 inhibition, did not promote OPIDP whereas the effect of 90 mg/kg, sc (corresponding to about 50-60% inhibition) was mild and not statistically significant. OPIDP induced by DBDCVP (5 mg/kg, sc) in rats was promoted by molinate (100 mg/kg, sc). In conclusion, protection from DBDCVP neuropathy by molinate is correlated with inhibition of NTE whereas promotion of DBDCVP neuropathy is associated with > 50% M200 inhibition.     

The relationship between isofenphos cholinergic toxicity and the development of polyneuropathy in hens and humans

Species differences have been observed between hen and human clinical manifestations of isofenphos toxicities. Hens treated with the insecticide isofenphos (90 mg/kg p.o.) developed severe cholinergic toxicity followed by mild organophosphate-induced delayed polyneuropathy (OPIDP). However, a patient developed severe OPIDP, which was preceded by very mild cholinergic signs, after an attempted suicide with a commercial formulation containing isofenphos and phoxim, an insecticide not causing OPIDP (estimated doses were 500 and 125 mg/kg, respectively). To explain this difference the following hypotheses were tested: (1) phoxim is a promoter of isofenphos-induced OPIDP; (2) whereas neuropathy target esterase (NTE) is thought to be the target of OPIDP, activation of isofenphos by liver microsomes causes the formation of more potent NTE inhibitor(s) in humans than in hens; (3) in contrast to hen NTE, the sensitivity of the human enzyme to such inhibitor(s) is higher than that of acetylcholinesterase (AChE), the target of cholinergic toxicity. Results showed that phoxim (22.5 mg/kg p.o.) was not a promoter of OPIDP in hens and that the ratio AChE inhibition:NTE inhibition by microsome-activated isofenphos was similar for both hen and human enzymes. The schedule of antidotal treatment in hens is the likely explanation for the observed difference from the patient. Peak AChE inhibition was maintained in hen brain up to 6 days after a single dose of isofenphos, suggesting prolonged pharmacokinetics. However, the AChE reactivator pyridine-2-aldoxime (2-PAM) was given to hens before isofenphos and then every 8 h, whereas continuous 2-PAM infusion was provided to the patient. When 2-PAM was given to hens every hour after isofenphos (90 mg/kg p.o.), the birds remained asymptomatic. Since other organophosphates may have a prolonged pharmacokinetics, testing procedures for the potential of these insecticides to cause OPIDP may underestimate the risk for humans.     

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.