Inhibitor Report for: Dibutyl-dichlorovinyl-phosphate~DBVP

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

Neuropathy target esterase (neurotoxic esterase, NTE), a protein thought to be involved in the production of organophosphorus compound-induced delayed neurotoxicity (OPIDN), has been postulated to be a component of endogenous neuronal protein phosphorylation systems. The purpose of this work was to test this hypothesis as well as to investigate further the role of endogenous protein phosphorylation in toxic neuropathies. White Leghorn hens were dosed with the neuropathic compounds di-1-butyl-2,2-dichlorovinyl phosphate (dibutyl dichlorvos, DBDCV), tri-o-cresyl phosphate (TOCP), or acrylamide, and regions from brain were fractionated into axolemmal, synaptosomal, and microsomal preparations. Radiolabeling of NTE or endogenously phosphorylated proteins was carried out by incubation with [14C]-DFP or gamma-[32P]-ATP, respectively. Radiolabeled proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography. Relative amounts of phosphoproteins were quantified by densitometry of the autoradiographs. Changes in endogenous phosphorylation of a protein exhibiting the characteristics of NTE were not observed in these experiments. However, levels of a [32P]-labeled 50-kDa brainstem axolemmal protein were decreased significantly on d 15, but not on d 1, 3, 7, or 10 after dosing with 2.8 mg/kg DBDCV. Clinical signs of ataxia and histopathological findings of axonal degeneration in the spinocerebellar tracts of the brainstem were evident on d 10-15, and hens were unable to perch on a horizontal wooden rod from d 12 after dosing with DBDCV. The decrease in the 50-kDa phosphoprotein was not observed on d 15 after the production of clinically evident neuropathy with either 14 daily doses of 50 mg/kg acrylamide or with a single dose of 500 mg/kg TOCP. These results suggest that NTE is not an endogenously phosphorylated protein under the conditions of these experiments. However, an effect on endogenous phosphorylation limited to a 50-kDa axolemmal protein was selectively produced by treatment with a neuropathic dose of DBDCV that was in evidence only after clinical signs and histopathological findings of axonopathy were apparent.

Carboxylesterases (CbxE) can be inhibited by organophosphorus esters (OPs) without causing clinical evidence of toxicity. CbxE are thought to protect the critical enzyme acetylcholinesterase (AChE) from OP inhibition in animals. CbxE and AChE are both present in neuroblastoma cells, but, even though these cells have potential to be an in vitro model of OP toxicity, the effect of OPs on CbxE and the relationship of CbxE inhibition and AChE inhibition have not yet been examined in these cells. Therefore, this study examined concentration-related OP-induced inhibition of CbxE in human SH-SY5Y and mouse NB41A3 neuroblastoma cells with 11 active esterase inhibitors: paraoxon, malaoxon, chlorpyrifos-oxon, tolyl saligenin phosphate (TSP), phenyl saligenin phosphate (PSP), diisopropyl phosphorofluoridate (DFP), mipafox, dichlorvos, trichlorfon, dibutyryl dichlorovinyl phosphate (DBVP), and dioctyl dichlorovinyl phosphate (DOVP). All could inhibit CbxE, although the enzyme was less likely to be inhibited than AChE following exposure to 9 of the test compounds in the human cell line and to all 11 of the test compounds in the murine cell line. Species differences in concentration-related inhibitions of CbxE were evident. When cells were exposed first to an OP with a low IC50 toward CbxE (PSP), followed by an OP with high affinity for AChE (paraoxon or malaoxon), inhibitions of CbxE and AChE were additive. This indicated that CbxE did not protect AChE from OP-induced inhibition in this cell culture model.

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

Neuropathy target esterase (neurotoxic esterase, NTE), a protein thought to be involved in the production of organophosphorus compound-induced delayed neurotoxicity (OPIDN), has been postulated to be a component of endogenous neuronal protein phosphorylation systems. The purpose of this work was to test this hypothesis as well as to investigate further the role of endogenous protein phosphorylation in toxic neuropathies. White Leghorn hens were dosed with the neuropathic compounds di-1-butyl-2,2-dichlorovinyl phosphate (dibutyl dichlorvos, DBDCV), tri-o-cresyl phosphate (TOCP), or acrylamide, and regions from brain were fractionated into axolemmal, synaptosomal, and microsomal preparations. Radiolabeling of NTE or endogenously phosphorylated proteins was carried out by incubation with [14C]-DFP or gamma-[32P]-ATP, respectively. Radiolabeled proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography. Relative amounts of phosphoproteins were quantified by densitometry of the autoradiographs. Changes in endogenous phosphorylation of a protein exhibiting the characteristics of NTE were not observed in these experiments. However, levels of a [32P]-labeled 50-kDa brainstem axolemmal protein were decreased significantly on d 15, but not on d 1, 3, 7, or 10 after dosing with 2.8 mg/kg DBDCV. Clinical signs of ataxia and histopathological findings of axonal degeneration in the spinocerebellar tracts of the brainstem were evident on d 10-15, and hens were unable to perch on a horizontal wooden rod from d 12 after dosing with DBDCV. The decrease in the 50-kDa phosphoprotein was not observed on d 15 after the production of clinically evident neuropathy with either 14 daily doses of 50 mg/kg acrylamide or with a single dose of 500 mg/kg TOCP. These results suggest that NTE is not an endogenously phosphorylated protein under the conditions of these experiments. However, an effect on endogenous phosphorylation limited to a 50-kDa axolemmal protein was selectively produced by treatment with a neuropathic dose of DBDCV that was in evidence only after clinical signs and histopathological findings of axonopathy were apparent.

Carboxylesterases (CbxE) can be inhibited by organophosphorus esters (OPs) without causing clinical evidence of toxicity. CbxE are thought to protect the critical enzyme acetylcholinesterase (AChE) from OP inhibition in animals. CbxE and AChE are both present in neuroblastoma cells, but, even though these cells have potential to be an in vitro model of OP toxicity, the effect of OPs on CbxE and the relationship of CbxE inhibition and AChE inhibition have not yet been examined in these cells. Therefore, this study examined concentration-related OP-induced inhibition of CbxE in human SH-SY5Y and mouse NB41A3 neuroblastoma cells with 11 active esterase inhibitors: paraoxon, malaoxon, chlorpyrifos-oxon, tolyl saligenin phosphate (TSP), phenyl saligenin phosphate (PSP), diisopropyl phosphorofluoridate (DFP), mipafox, dichlorvos, trichlorfon, dibutyryl dichlorovinyl phosphate (DBVP), and dioctyl dichlorovinyl phosphate (DOVP). All could inhibit CbxE, although the enzyme was less likely to be inhibited than AChE following exposure to 9 of the test compounds in the human cell line and to all 11 of the test compounds in the murine cell line. Species differences in concentration-related inhibitions of CbxE were evident. When cells were exposed first to an OP with a low IC50 toward CbxE (PSP), followed by an OP with high affinity for AChE (paraoxon or malaoxon), inhibitions of CbxE and AChE were additive. This indicated that CbxE did not protect AChE from OP-induced inhibition in this cell culture model.

Young animals are resistant to organophosphate-induced delayed neuropathy (OPIDP), although biochemical changes on Neuropathy Target Esterase (NTE) caused by neuropathic organophosphorus esters (OP) are similar to those observed in the sensitive hen. We report here that the resistance of chicks to single doses of neuropathic OPs is not absolute because ataxia was produced in 40-day-old chicks by 2,2-dichlorovinyl dibutyl phosphate (DBDCVP, 5.0 or 10.0 mg/kg s.c.) and by diisopropyl phosphorofluoridate (DFP, 2.0 mg/kg s.c.). However, the clinical picture was different from that usually seen in hens; spasticity and complete recovery being the main features. alpha-Tolyl sulphonyl fluoride (PMSF, 300 mg/kg s.c.) promoted both DBDCVP neuropathy (5.0 or 10.0 mg/kg s.c.) and non-neuropathic doses of DFP (1.5 mg/kg s.c.) or DBDCVP (1.0 mg/kg s.c.). The lowest promoting dose of PMSF given 24 hr after 1.5 mg/kg of DFP was 30 mg/kg. Higher doses had a more severe effect but no further increase of OPIDP severity was obtained with doses ranging from 90 to 300 mg/kg. PMSF (30 mg/kg) protected 40-day-old chicks from subsequent doses of neuropathic OPs even when a promoting dose of PMSF followed. At 60 days of age, chicks' resistance to OPIDP decreased because lower doses of neuropathic OPs became effective and, similarly to hens, PMSF did not fully protect from subsequent promotion. In 40-day-old chicks the threshold of NTE inhibition for OPIDP development was 95-97% (DBDCVP 5.0 mg/kg). When promotion followed initiation, the minimal effective inhibition of NTE for initiation by neuropathic OPs was about 90%. In 36-day-old chicks, PMSF (300 mg/kg) promoted OPIDP when given up to 5 days after DFP (1.5 mg/kg) when residual NTE inhibition in brain and sciatic nerve was about 40%. We conclude that chicks' resistance to OPIDP might reflect either a less effective initiation by phosphorylated NTE or a more efficient repair mechanism or both, and also that promotion is likely to involve a target other than NTE.

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.

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.

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 (ka = 5.4 x 10(6)) relative to that from spinal cord (ka = 13.9 x 10(6)) or brain (ka = 20.6 x 10(6)). 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous work in our laboratory indicated that di-n-butyl-2,2-dichlorovinyl phosphate (DBCV) produced electrophysiologic changes in hen peripheral nerve that coincided with the development of histopathologic changes and neurologic signs of peripheral neuropathy. The purpose of the present study was to follow the time course for the development of the electrophysiologic changes and to determine whether pretreatment with the phosphinate analog of DBCV (DBCV-P), a nonageable organophosphorus compound, prevented these effects. Although significant electrophysiologic deficits occurred in the tibial and sciatic nerve 24 h after DBCV treatment, the most marked changes coincided with the onset of clinical signs of organophosphorus-induced delayed neuropathy (14-21 d). The sciatic and tibial nerves were equally susceptible to DBCV in producing deficits characterized by changes in the relative refractory period and an increased strength-duration threshold. Pretreatment with DBCV-P prevented the clinical signs and also attenuated the electrophysiologic deficits induced by DBCV treatment. These data suggest that electrophysiologic deficits occur before clinical signs of organophosphorus-induced delayed neuropathy (OPIDN) and may be indicative of a link between neurotoxic esterase (NTE) inhibition and onset of overt clinical toxicity.

The induction of central-peripheral distal axonopathy in hens singly dosed with some organophosphorus (OP) compounds, such as di-n-butyl-2,2-dichlorovinyl phosphate (DBDCVP), requires greater than 80% organophosphorylation and subsequent intramolecular rearrangement ("aging") of a protein [neuropathy target esterase (NTE)] in the axon. Suprathreshold biochemical reaction, 24 h after dosing with DBDCVP (0.75-1.00 mg/kg s.c.), is shown to be associated with progressive decrement of retrograde axonal transport in sensory and motor fibers. The maximum transport deficit (about 70% reduction) is reached 7 days after DBDCVP, prior to the appearance of axonal degeneration and the onset of clinical signs of neuropathy (day 10-11). By contrast, phenylmethylsulfonyl fluoride (30 mg/kg s.c.), an agent that prevents the development of OP neuropathy by inhibiting NTE without the "aging" reaction, had no effect on axon transport, nerve fiber integrity, or clinical status and, when administered prior to a neurotoxic dose of DBDCVP (1.00 mg/kg s.c.), prevented DBDCVP effects. Paraoxon (0.2 mg/kg s.c.) neither inhibited NTE nor caused deficits in retrograde transport or neuropathy. Taken in concert, these studies demonstrate that induced deficits in retrograde transport are associated with the pathogenesis of OP-induced nerve-fiber degeneration and the threshold-initiating mechanism thereof.