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     

Age sensitivity to organophosphate-induced delayed polyneuropathy. Biochemical and toxicological studies in developing chicks

Young animals are resistant to organophosphate-induced delayed polyneuropathy (OPIDP). The putative target protein in the nervous system for initiation of OPIDP in the adult hen is an enzyme called Neuropathy Target Esterase (NTE), which is dissected by selective inhibitors among nervous tissue esterases hydrolysing phenyl valerate (PV). We report here that the pool of PV-esterases sensitive to paraoxon was different in peripheral nerves of chicks as compared to that of hens while that of brain and spinal cord was not. NTE activity decreased with age in brain, spinal cord and peripheral nerve, but its sensitivity to several inhibitors remained unchanged. In the adult hen more than 70% inhibition of peripheral nerve NTE by neuropathic OPs is followed by deficit of retrograde axonal transport, axonal degeneration and paralysis. Similar NTE inhibition in 40-day-old or younger chicks however is not followed by changes in retrograde axonal transport nor by OPIDP. Chicks aged 60 to 80 days are only marginally sensitive to a single dose of DFP otherwise clearly neuropathic to hens. In vitro and in vivo phosphorylation by DFP and subsequent aging of brain NTE is similar both in chicks and in hens. The recovery of NTE activity monitored in vivo after inhibition by DFP is faster (half-life of about 3 days) in chick peripheral nerves as compared to chick brain, hen brain and hen peripheral nerve (half-life of about 5 days). It is concluded that the reduced sensitivity to OPIDP in chicks is not due to differences in OP-NTE interactions. The resistance might be explained by a more efficient repair mechanism, as suggested by the faster recovery of peripheral nerve NTE activity.     

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.     

Clinical expression of organophosphate-induced delayed polyneuropathy in rats.

Single doses of certain organophosphates (OP), such as dibutyl-2,2-dichlorovinyl phosphate (DBDCVP) cause organophosphate-induced delayed polyneuropathy (OPIDP) in hens. Clinical effects correlate with inhibition of neuropathy target esterase (NTE) which is considered the target for this toxicity. Pre-treatment with non-neuropathic NTE inhibitors, such as phenylmethanesulfonyl fluoride (PMSF), protects from OPIDP. However, when given after OPs, these compounds promote OPIDP. Chicks are relatively resistant to OPIDP despite high NTE inhibition. It has also always been reported that rats represent a species which is resistant to OPIDP and that they might develop morphological but not clinical signs of OPIDP. We report here that clinical OPIDP can be produced in 3.5- and 6-month-old rats by DBDCVP (5 mg/kg s.c.) and that it correlates with high (> 90%) NTE inhibition. When PMSF (120 mg/kg s.c. x 2) was given after DBDCVP, OPIDP was promoted. Pretreatment with PMSF protected from OPIDP. We conclude that resistance to OPIDP in the rat is age-related, as it is in the hen.     

Anomalous biochemical responses in tests of the delayed neuropathic potential of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved isomers and of some higher O-alkyl homologues

The interaction with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) in vivo of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved stereoisomers and five higher O-alkyl homologues has been examined along with the ability of these compounds to cause organophosphorus-induced delayed polyneuropathy (OPIDP) in adult hens. For the lower homologues AChE was more sensitive than NTE and it was impossible to achieve high inhibition of NTE in vivo without both prophylaxis and therapy against acute anticholinesterase effects; for then-hexyl homologue high inhibition of NTE could be achieved without obvious anticholinesterase effects and spontaneous reactivation of inhibited AChE was seen as in vitro. The maximum tolerated dose ofl(–) methamidophos or of the ethyl oriso-propyl homologues did not inhibit NTE more than 60%, and surviving birds did not develop OPIDP. Then-propyl,n-butyl andn-hexyl compounds caused typical OPIDP at doses causing a peak of 70–95% inhibition of NTE in brain, spinal cord and sciatic nerve soon after dosing. Racemic methamidophos caused unusually mild OPIDP associated with very high inhibition of NTE at doses estimated to be >8 times the unprotected LD₅₀ and thed-(+) isomer caused OPIDP at about 5–7× LD₅₀. Clinical effects correlated with histopathology in 19 out of 20 examined birds. In contrast to results of many previous studies with organophosphates and phosphonates, all these cases of OPIDP were associated with formation of inhibited NTE which could be reactivated ex vivo by treatment of autopsy tissue with KF solution. It is not clear whether aging of inhibited NTE had occurred but with less associated stabilisation of the enzyme-phosphorus bond or whether, even without aging, the unusual N-unsubstituted phosphoramidate caused sufficient disturbance in or near the NTE target to initiate the same degenerative process as that caused typically by generation of aged organophosphorylated NTE.     

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     

Neurotoxic esterase in peripheral nerve: Assay,inhibition, and rate of resynthesis

The potential neurotoxicity of organophosphate esters is usually evaluated by measuring neurotoxic esterase (NTE) inhibition in brains taken from dosed birds. An improved method to measure NTE in peripheral nerve has been developed and used to compare brain, spinal cord, and peripheral nerve NTE inhibition both in vitro and in vivo after a single dose of several organophosphates. Brain and spinal cord NTE activities are a good mirror of NTE activity of the sciatic nerve. The rate of resynthesis of NTE in peripheral nerves after inhibition with an effective dose of a neurotoxic organophosphate was similar to that in other nervous tissues.     

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.     

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.     

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.     

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     

An alternative in vitro method for detecting neuropathic compounds based on acetylcholinesterase inhibition and on inhibition and aging of neuropathy target esterase (NTE)

Organophosphorus-induced delayed polyneuropathy (OPIDP) is a syndrome induced by certain organophosphorus compounds (OPs) through a mechanism based on the inhibition and further modification (aging) of neuropathy target esterase (NTE). OECD guidelines for testing the capability of OPs to trigger OPIDP include two in vivo tests with hens. Activities of acetylcholinesterase and NTE found in SH-SY5Y human neuroblastoma cells were inhibited by 10 different OPs with kinetics similar to those found with chicken brain enzymes (model system for in vivo and in vitro–ex vivo assays). NTE in SH-SY5Y cells inhibited by these OPs aged and reactivated similarly to that described for hen brain NTE ex vivo. In short, we have developed an alternative methodology for predicting the capability of OPs to induce OPIDP based on the inhibition kinetics of acetylcholinesterase and NTE and on the capability of OPs to age the inhibited NTE from SH-SY5Y cell line. The results obtained always agreed with the previously reported ex vivo results with hen brain. The developed methodology correctly predicted the neuropathic potential of the tested OPs in eight cases. The in vivo–in vitro discrepancies with two of the tested compounds can be explained on the basis of differences between in vivo and in vitro biotransformation.     

The anomalous behaviour of dimethyl phosphates in the biochemical test for delayed neurotoxicity

Several dimethyl phosphate behave anomalously in tests for delayed neurotoxicity. Doses given to hens caused high inhibition of brain neurotoxic esterase (NTE) but no ataxia. Less inhibition of NTE was seen in spinal cord than in brain. Di-isopropyl phosphorofluoridate caused equal inhibition of NTE in brain and cord. When dosing with dimethyl phosphates was repeated NTE inhibition in cord increased and pair-dosed birds became ataxic. In vitro brain and cord NTE were indistinguishable but the in vivo discrepancy between inhibition of brain and cord NTE was matched by a similar discrepancy in inhibition of AChE. It appears that ataxia arises from inhibition of spinal cord NTE and that only in the present cases (among about 200) was the effect in brain not a perfect biochemical monitor.     

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.     

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.     

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.     

The influence of chirality on the delayed neuropathic potential of some organophosphorus esters: neuropathic and prophylactic effects of stereoisomeric esters of ethyl phenylphosphonic acid (EPN oxon and EPN) correlate with quantities of aged and unaged neuropathy target esterase in vivo

Organophosphate-induced delayed polyneuropathy (OPIDP) is thought to result from organophosphorylation of neuropathy target esterase (NTE), followed by an "aging" of the phosphorylated NTE. Prophylactic against OPIDP should thus be achieved by production of an inhibited but "nonaging" NTE. Resolved stereoisomers of ethyl phenylphosphonic acid esters produce two forms of inhibited NTE; in vitro one form ages rapidly and the other only negligibly. The present study examined the in vivo effects of two preparations of incompletely resolved isomers of EPN oxon (ethyl 4-nitrophenyl phenylphosphonate) and its thionate on adult hen brain and spinal cord NTE and the relationship of inhibition and aging to the development of OPIDP. Single doses of the L-(-)-isomers (Preparation A, 7:3 proportion of isomers, or Preparation B, 9:1) caused severe neuropathy after doses which produced 70% aged inhibited NTE and mild effects after 50-60%. Single doses of the D-(+)-isomers produced either equal amounts of aged and unaged inhibited NTE (Preparation A) or predominantly unaged (Preparation B): the amount of aged was never more than 50% and no clinical OPIDP occurred. Doses of D-(+) which produced 50% unaged inhibited NTE were protective: challenge with the highly neuropathic phenyl saligenin cyclic phosphate did not cause OPIDP. All effects are consistent with the two-stage initiation process which requires both inhibition of NTE and subsequent modification of the protein by an "aging" process. Previously reported neuropathic effects of D-(+)-EPN probably reflect a substantial proportion of L-(-)-isomer present in the test material. Neuropathic studies with chiral OP esters should consider the possibility of production of protective unaged inhibited NTE in test animals.     

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.     

Biochemical and clinical tests of the delayed neuropathic potential of some O-alkyl O-dichlorophenyl phosphoramidate analogues of methamidophos (O,S-dimethyl phosphorothioamidate)

The interaction in vivo of four O-alkyl O-2,5-dichlorophenyl phosphoramidates with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) and their ability to cause delayed polyneuropathy in hens has been examined. Previous studies in vitro (Vilanova, Johnson & Vicedo, Pestic. Biochem. Physiol., 28 (1987) 224) had led to the prediction that these compounds would not be neuropathic but, rather, would be prophylactic agents against organophosphorus-induced delayed polyneuropathy. In vivo the effects of these esters on the enzymes differ in 2 respects from effects in vitro: (i) Relative sensitivity of the enzymes was different: thus greater than 50% of brain NTE remained 24 h after an oral dose of 15 mg/kg of the n-hexyl ester while only 10-30% of AChE remained although NTE was the more sensitive enzyme in vitro; (ii) In no case could the inhibited NTE or AChE in autopsy samples from birds dosed with any of the 4 esters be reactivated by treatment with potassium fluoride in vitro: the inhibited enzymes produced by incubation of tissue with the esters in vitro had been reactivatable. Prophylaxis, with therapy in some cases, was required to prevent acute anticholinesterase poisoning when doses were sufficient to cause high inhibition of neural NTE. Inhibition in brain was typically 5-10% more than in spinal cord and 10-15% more than in sciatic nerve. Unambiguous signs of polyneuropathy (Grade 3 or more on an 8-point scale) were not seen in birds observed up to 3 weeks after doses which caused less than 70% inhibition of NTE in brain and spinal cord or less than 60% inhibition in sciatic nerve of pair-dosed birds assayed 24 h after dosing. Doses of 300, 10, 100 and 65 mg/kg, respectively, of the methyl, ethyl, n-butyl and n-hexyl esters caused greater than 70% inhibition of NTE in all 3 neural tissues and neuropathy in the majority of observed birds. Analysis of consolidated dose/response data from 36 assayed and 51 observed birds showed that effects of Grade 3 or more were produced in about 90% of birds when inhibition of NTE was greater than 90% in brain, greater than 85% in spinal cord or greater than 75% in sciatic nerve.(ABSTRACT TRUNCATED AT 400 WORDS)