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.     

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.     

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.     

Local application of neuropathic organophosphorus compounds to hen sciatic nerve: inhibition of neuropathy target esterase and peripheral neurological impairments.

Diisopropyl phosphorofluoridate (DFP), mipafox, cresylsaligenyl phosphate, and phenylsaligenyl phosphate were applied to a 1.5-cm segment of the common trunk of the sciatic nerve in adult hens. At doses of 18-182 micrograms mipafox and 9-110 micrograms DFP, inhibition of neuropathy target esterase (NTE) for the treated segment was over 80%, whereas for the adjacent distal and proximal segments inhibition was under 40%, 15 min after application. NTE was not affected in the peripheral distal terminations arising from the common sciatic nerve (peroneal branches), contralateral sciatic nerve, brain, and spinal cord. A 24-hr study suggested a displacement of the activity-free region toward more distal segments of the nerve. All animals treated with 55 and 110 micrograms DFP or 110 micrograms mipafox lost a characteristic avian retraction reflex in the treated leg 9-15 days after dosing, suggesting peripheral neurological alterations. Only hens dosed at the maximum dose in both extremities presented alterations in motility (Grade 1 or 2 on a 0-8 scale), suggesting no significant central nervous system alterations. Electron microscopy of peroneal branches showed axon swelling and accumulation of smooth endoplasmic reticulum similar to animals dosed systemically (s.c.) with 1-2 mg/kg DFP. The branches also contained granular and electron-dense materials, as well as some intraaxonal and intramyelinic vacuolization. Clinical effects were not observed in animals protected with a 30 mg/kg (s.c.) dose of phenylmethanesulphonyl fluoride. It is concluded that the peripheral neurological effects of local dosing correlate with the specific modification of NTE in a segment of sciatic nerve and that the axon is a more likely target than the perikaryon or nerve terminal in the triggering mechanism of this axonopathy.     

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     

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.     

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.     

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.     

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 correlation between the recovery rate of neurotoxic esterase activity and sensitivity to organophosphorus-induced delayed neurotoxicity

Neurotoxic esterase (NTE) has been proposed to be the initiation site of organophosphorus compound-induced delayed neurotoxicity (OPIDN). There are two apparent problems associated with this hypothesis: NTE activity in the brain returns to nearly normal levels before the onset of the neuropathy, and NTE is present in and inhibited by organophosphorus compounds in young animals and other species which are relatively insensitive to the neurotoxic effects of these compounds. This paper presents data suggesting that differences in the recovery rates of NTE activity may account for some of these discrepancies. First, the onset of recovery of NTE activity following sc administration of 1.7 mg/kg of O,O-diisopropylphosphorofluoridate (DFP) in the hen sciatic nerve occurred several days later than in the brain. Furthermore, recovery was slower in distal than proximal parts of the nerve. This information indicates that NTE activity is depressed for a longer period at the site of the neuropathy than it would appear from the measurement of NTE activity in brain. Second, the rate of recovery of NTE activity was faster in the brains of chicks, of rats, and of hens treated with a daily po dose of 15 mg/kg cortisone acetate than it was in untreated hens. However, there was no significant increase in the NTE recovery rate in the peripheral nerves of the chicks or the cortisone-treated hens. Thus, it appears that although slower distal recovery could account for the greater sensitivity of longer axons to OPIDN, other factors are operating in chicks and cortisone-treated hens.     

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     

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.     

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.     

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.     

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.     

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

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

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     

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.     

An electrophysiologic and ultrastructural study of the phenylmethanesulfonyl fluoride protection against a delayed organophosphorus neuropathy

The delayed organophosphorus neuropathy caused by diisopropylfluorophosphate (DFP) can be prevented by pretreatment with phenylmethanesulfonyl fluoride (PMSF). A single injection of DFP (2 mg/kg) into a cat femoral artery produced a delayed neuropathy in the injected leg. Clinical neurotoxic signs in the DFP treated leg were most prominent at 21 to 28 days after DFP administration: a high-step gait with some tip-toe walking. During that time the capacity of the cat soleus alpha-motor nerve terminals to generate a stimulus-evoked repetitive discharge, known as SBR, was greatly attenuated. At that time, the ultrastructure of the motor nerve terminals demonstrated prominent alterations that correlated well with the motor nerve terminal SBR deficit. These alterations included the presence of extensive whorls in nerve terminals and axoplasms, the retraction and disruption of nerve terminals from the synaptic cleft, and a widening of secondary junctional folds. From the sampled population, the incidence of normal terminals in soleus muscles of the DFP-treated leg was only 2%. Cats which received PMSF (30 mg/kg ip) 24 hr before DFP administration did not develop any neurotoxic signs. Motor movements were normal. The SBR function of the soleus alpha-motor nerve terminals was not lost and its incidence approached normal values. Moreover, the ultrastructure was normal in 86% of examined neuromuscular junctions in the PMSF pretreated DFP cats. Thus, in this model, pretreatment with PMSF protected cats against the delayed neurotoxic effects of organophosphorus poisoning.     

The effects of phenylmethanesulfonyl fluoride on delayed organophosphorus neuropathy

A delayed localized neuropathy of peripheral nerves in a single hind leg of the cat develops after a single intraarterial 2 mg/kg injection of diisopropylfluorophosphate (DFP). This neuropathy is manifested by a maximum loss of the capacity of soleus -motor nerve terminals to generate stimulus-bound repetition 21 days after DFP exposure. Phenylmethanesulfonyl fluoride (PMSF) is a protective inhibitor of the neurotoxic esterase which is associated with the development of the delayed organophosphorus neuropathy. Pretreatment of cats with PMSF (30 mg/kg i.p.) 24 h before the DFP injection protected the cats from the delayed neuropathy. No clinical neurotoxic signs were observed at 21 days after DFP. The stimulus-bound repetitive capacity of soleus -motor nerve terminals was not lost at this time and its incidence was much greater than that which occurred in cats not pretreated with PMSF.This work was supported by U.S. Public Health Service National Institute for Neurological and Communicative Diseases and Stroke Grants NS-01447 and NS-11948