Phenylmethylsulfonyl fluoride protects against the degradation of neurofilaments in tri-ortho-cresyl phosphate (TOCP) induced delayed neuropathy

Tri-ortho-cresyl phosphate (TOCP) is an organophosphorus ester, which can cause a type of neurotoxicity known as organophosphate-induced delayed neuropathy (OPIDN). Our recent study has shown that the enhanced degradation of neurofilament (NF) in peripheral nerve of hens is an early event of TOCP-induced OPIDN (Song et al., 2009). The main objective of this investigation is to study the effect of TOCP administration on NF content and NF degradation when OPIDN is blocked by pretreatment with phenylmethylsulfonyl fluoride (PMSF). The hens were pretreated 24 h earlier with PMSF and subsequently treated with a single dosage of 750 mg/kg TOCP, then sacrificed on the corresponding time points of 0, 1, 5, 10, and 21 days after dosing TOCP, respectively. The tibial nerves were dissected, homogenized, and centrifuged at 100,000 × g. The level of NF triplet protein in both pellet and supernatant fractions of tibial nerves was determined. Western blotting analysis showed a significant increase of three NF subunits in hens treated with PMSF and TOCP compared with the control. These changes were observed within 24 h of PMSF administration and then followed by an obvious recovery. Furthermore, accompanied with the increase of NF content, a significant decline in NF-L degradation rate was observed in both fractions of tibial nerves. Taken together, these results demonstrated the pretreatment with PMSF could inhibit TOCP-induced NF degradation while it protected hens against the development of OPIDN, which suggested the inhibition of NF-associated protease in peripheral nerves might be an underlying protective mechanism of PMSF against OPIDN.     

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.     

三邻甲苯磷酸酯暴露鸡脊髓组织中Cofilin-1b的差异表达

目的从三邻甲苯磷酸酯(TOCP)暴露鸡的脊髓组织中筛选可能与调控微丝解聚作用相关的差异表达蛋白,为探讨有机磷化合物诱发的迟发性神经毒性(OPIDN)作用机制提供靶蛋白依据。方法 42只罗曼鹤母鸡随机分成1000 mg/kg TOCP组、预先给予40 mg/kg苯甲基磺酰氟(PMSF)后再投1000 mg/kg TOCP的干预组和生理盐水对照组,每组14只。染毒第5和20天,每组分别处死4只鸡,低温环境下分离脊髓,提取总蛋白。利用双向电泳结合质谱分析技术,筛选和鉴定可能与调控微丝解聚作用相关的差异表达蛋白。结果 TOCP组鸡于染毒第7日前后出现进行性共济失调和肌无力等OPIDN典型症状,起病从下肢远端部分开始且病变程度随时间逐渐加重直至全瘫,而其他组鸡在实验观察期间未见OPIDN症状。TOCP组鸡于暴露第5天,分别与对照组和PMSF前干预组比较,其脊髓组织肌动蛋白解聚因子Cofilin-1b分别下调3.4倍和2.8倍,且有统计学意义(差异表达<0.5或差异表达>2),而PMSF前干预组与对照组比较,鸡脊髓组织Cofilin-1b的表达差异无统计学意义。在TOCP暴露第20天,TOCP组鸡脊髓组织Cofilin-1b表达与其他两组比较尽管有下降趋势,但无显著性变化。结论 TOCP暴露能导致鸡脊髓神经组织Cofilin-1b表达在早期显著下调,且该蛋白表达下调可能与微丝骨架结构紊乱及其OPIDN诱发机制有关。     

The homeostasis of phosphatidylcholine and lysophosphatidylcholine was not disrupted during tri-o-cresyl phosphate-induced delayed neurotoxicity in hens

Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity (OPIDN) except for the essential inhibition of neuropathy target esterase (NTE). We hypothesized that the homeostasis of lysophosphatidylcholine (LPC) and/or phosphatidylcholine (PC) in nervous tissues might be disrupted after exposure to the organophosphates (OP) which participates in the progression of OPIDN because new clues to possible mechanisms of OPIDN have recently been discovered that NTE acts as lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. To bioassay for such phospholipids, we induced OPIDN in hens using tri-o-cresyl phosphate (TOCP) as an inducer with phenylmethylsulfonyl fluoride (PMSF) as a negative control; and the effects on the activities of NTE, LysoPLA and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE enzyme in the brain, spinal cord, and sciatic nerves were examined. The results demonstrated that the activities of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB and PLB were significantly inhibited in both TOCP- and PMSF-treated hens. The inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient in the nervous tissues. Moreover, the NTE inhibited by TOCP was of the aged type, while nearly all of the NTE inhibited by PMSF was of the unaged type. No significant change in PC or LPC levels was observed, while the GPC level was significantly decreased. However, there is no relationship found between the GPC level and the delayed symptoms or aging of NTE. All results suggested that LPC and/or PC homeostasis disruption may not be a mechanism for OPIDN because the PC and LPC homeostasis was not disrupted after exposure to the neuropathic OP, although NTE, LysoPLA, and PLB were significantly inhibited and the GPC level was remarkably decreased.     

Identification of differentially expressed proteins related to organophosphorus‐induced delayed neuropathy in the brains of hens

Some organophosphorus compounds can cause organophosphate‐induced delayed neuropathy (OPIDN). Incidents have been documented for decades, however, little is known about which proteins contribute to the initiation, progression and development of OPIDN. In this study, 51 hens were divided into three groups. The tri‐ortho‐cresyl‐phosphate (TOCP) group was treated with 1000 mg kg^–1 TOCP whereas the control group was treated with an equivalent volume of vehicle. The PMSF + TOCP group was treated subcutaneously with 40 mg kg^–1 phenylmethylsulfonyl fluoride (PMSF), followed by 1000 mg kg^–1 TOCP 24 h later. Proteins in the brains of hens were separated by two‐dimensional polyacrylamide gel electrophoresis on day 5 after TOCP administration. Mass spectrometry identified eight differentially expressed proteins. Among these proteins, downregulated expression of glutamine synthetase (GS) in the brains of hens after TOCP treatment was further confirmed by real time RT‐PCR and ELISA. Moreover, the brains of hens exposed to TOCP exhibited increased levels of glutamate (Glu) and cytosolic calcium concentration ([Ca²⁺]_i), and a decreased level of glutamine (Gln). However, there were no significant differences in GS expression or levels of Glu, Gln, and [Ca²⁺]_i in the brains of hens among the groups on day 21 after TOCP administration. These results indicate that TOCP exposure downregulates GS expression in the brains of hens, and that downregulation of GS is accompanied by increased levels of Glu and [Ca²⁺]_i in the early stage after TOCP administration. It is also suggested that the downregulated expression of GS might be associated with OPIDN through the disruption of homeostasis of the Glu–Gln cycle and [Ca²⁺]_i. Copyright © 2013 John Wiley & Sons, Ltd.     

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

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

Effects of calcium gluconate and PMSF in the treatment of acute intoxication of chicken by TOCP

To examine the efficacy of calcium gluconate (two doses of Ca-Glu 5 mg/kg i.v.) to alleviate the injurious effects of organophosphorus induced delayed neuropathy (OPIDN) in the presence or absence of phenylmethanesulfonyl fluoride (PMSF 90 mg/kg i.m.), 14 groups of four isabrown hens were used. To measure the lymphocyte neuropathy target esterase (LNTE)activity, groups receiving just distilled water (control), groups receiving just Tri-orto-cresyl phosphate (TOCP; 500 mg/kg p.o.) (Positive control), and other groups receiving TOCP and Ca-Glu or PMSF simultaneously or 12 hours later following intoxication by TOCP were used. They were sacrificed 12 and 24 hours after the administration of TOCP. To observe a 28-day time course of neurotoxicity scores and calcium plasma concentration, five groups were used. Regarding free Ca(2+)in the plasma, the positive control produced a characteristic profile time course up and down during 28 days, and some hens with maximum score of neurotoxicity in 28 days. The treatment, which prevented greater oscillation in free Ca(2+) in the plasma, presented a decrease in OPIDN in relation to the positive control. Twelve hours after the administration of TOCP, LNTE was 70-80% inhibited when compared with control, whereas the first decrease in the free Ca(2+) in the plasma was significantly different from the control only 24 hours after the administration of TOCP. In summary, the sooner the Ca-Glu is started, the less severe the neuropathy effects.     

The time course of protection from delayed neurotoxicity induced by tri-o-cresyl phosphate and O,O-diisopropyl phosphorofluoridate by phenyl methyl sulfonyl fluoride in chickens

The time dependence of the ability of phenyl methyl sulfonyl fluoride (PMSF) to protect adult hens from developing signs of paralysis following the administration of 750 mg/kg tri-ortho-cresyl phosphate (TOCP), p.o., of 1.7 mg/kg O,O-diisopropyl phosphorofluoridate (DFP), s.c., was investigated. PMSF was able to protect the hens from organophosphorus-induced delayed neurotoxicity (OPIDN) when given between 1 and 24 h before the administration of TOCP, or when given 4 h before DFP. However, PMSF was ineffective at preventing paralysis when given 24 h following dosing with TOCP or when given later than 4 h before DFP administration. These results support the notion that PMSF acts at the same site as the organophosphorus esters.     

Time-dependent changes of lipid peroxidation and antioxidative status in nerve tissues of hens treated with tri-ortho-cresyl phosphate (TOCP)

Tri-ortho-cresyl phosphate (TOCP) could induce a delayed neurodegenerative condition known as organophosphorus easter-induced delayed neurotoxicity (OPIDN) in human beings and sensitive animals. However, the mechanisms of OPIDN remain unknown. This study investigated the time-dependent changes of the lipid peroxidation (malondialdehyde, MDA) and antioxidative status (glutathione, GSH; glutathione peroxidase, GSH-Px; glutathione reductase, GR; superoxide dismutase, SOD and anti-reactive oxygen species, anti-ROS) in nerve tissues for elucidating the mechanism of OPIDN induced by TOCP. Adult hens were treated with TOCP by gavage at a single dosage of 750 mg/kg. TOCP was dissolved in corn oil and administered at 0.65 ml/kg. The control hens received an equivalent volume of corn oil by gavage. Hens were sacrificed after 0, 5, 10, 15 and 21 days of treatment and the cerebrum, spinal cord, sciatic nerve were dissected, homogenized and used for the determination of lipid peroxidation and antioxidative status. The results showed that treatment with TOCP increased lipid peroxidation and reduced the antioxidative status in cerebrum, spinal cord and sciatic nerve. The levels of MDA increased by 33% (P<0.01) in cerebrum on 5th day after TOCP treatment and at clinical sign score of 1-2, and increased respectively by 32% and 15% (P<0.01) in spinal cord and sciatic nerve on 10th day after TOCP treatment and at clinical sign score of 3-4. Further changes of MDA were also observed after 15 and 21 days post-dosing and at clinical sign score of 5-6 and 7-8. There is a decrease in the activities of SOD, GSH-Px, GR, anti-ROS, and GSH content in cerebrum, spinal cord and sciatic nerve of hens after 5, 10, 15 and 21 days post-dosing and at clinical sign score of 1-2, 3-4, 5-6 and 7-8. Thus, OPIDN induced by TOCP was associated with elevation of lipid peroxidation and reduction of antioxidative status, and the time-dependent changes of these indexes in hens nerve tissues occurred. Sciatic nerve was the main target tissue and MDA was most sensitive among all indexes. The time-dependent and tissue specific changes of lipid peroxidation and antioxidative status in cerebrum, spinal cord and sciatic nerve suggest that ROS and concomitant lipid peroxidation, at least in part, are involved in the toxic effects of TOCP on nerve tissues and that oxidative stress may play a role in the occurrence and development of OPIDN induced by TOCP.     

Changes of mitochondrial ultrastructures and function in central nervous tissue of hens treated with tri-ortho-cresyl phosphate (TOCP)

Tri-ortho-cresyl phosphate (TOCP), an organophosphorus ester, is capable of producing organophosphorus ester-induced delayed neurotoxicity (OPIDN) in humans and sensitive animals. The mechanism of OPIDN has not been fully understood. The present study has been designed to evaluate the role of mitochondrial dysfunctions in the development of OPIDN. Adult hens were treated with 750 mg/kg·bw TOCP by gavage and control hens were given an equivalent volume of corn oil. On day 1, 5, 15, 21 post-dosing, respectively, hens were anesthetized by intraperitoneal injection of sodium pentobarbital and perfused with 4% paraformaldehyde. The cerebral cortex cinerea and the ventral horn of lumbar spinal cord were dissected for electron microscopy. Another batch of hens were randomly divided into three experimental groups and control group. Hens in experimental groups were, respectively, given 185, 375, 750 mg/kg·bw TOCP orally and control group received solvent. After 1, 5, 15, 21 days of administration, they were sacrificed and the cerebrum and spinal cord dissected for the determination of the mitochondrial permeability transition (MPT), membrane potential (Δψ(m)) and the activity of succinate dehydrogenase. Structural changes of mitochondria were observed in hens' nervous tissues, including vacuolation and fission, which increased with time post-dosing. MPT was increased in both the cerebrum and spinal cord, with the most noticeable increase in the spinal cord. Δψ(m) was decreased in both the cerebrum and spinal cord, although there was no significant difference in the three treated groups and control group. The activity of mitochondrial succinate dehydrogenase assayed by methyl thiazolyl tetrazolium (MTT) reduction also confirmed mitochondrial dysfunctions following development of OPIDN. The results suggested mitochondrial dysfunction might partly account for the development of OPIDN induced by TOCP.     

Protein levels of neurofilament subunits in the hen central nervous system following prevention and potentiation of diisopropyl phosphorofluoridate (DFP)-induced delayed neurotoxicity(1).

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, which produces delayed neurotoxicity (OPIDN) in hens in 7-14 days. OPIDN is characterized by mild ataxia in its initial stages and severe ataxia or paralysis in about 3 weeks. It is marked by distal swollen axons, and exhibits aggregations of neurofilaments (NFs), microtubules, proliferated smooth endoplasmic reticulum, and multivesicular bodies. These aggregations subsequently undergo disintegration, leaving empty varicosities. Previous studies in this laboratory have shown an increased level of medium-molecular weight NF (NF-M) and decreased levels of high- and low-molecular weight NF (NF-H, NF-L) proteins in the spinal cord of DFP-treated hens. The main objective of this investigation was to study the effect of DFP administration on NF subunit levels when OPIDN is prevented or potentiated by pretreatment or post-treatment with phenylmethylsulfonyl fluoride (PMSF), respectively. Hens pretreated or post-treated with PMSF were killed 1, 5, 10, and 20 days after the last treatment. The alteration in NF subunit protein levels observed in DFP-treated hen spinal cords was not observed in protected hens. Estimation of NFs in the potentiation experiments, however, showed a different pattern of alteration in NF subunit levels. The results showed that an alteration in NF subunit levels in DFP-treated hens might be related to the development of OPIDN, since these changes were suppressed in PMSF-protected hens. However, results from PMSF post-treated hen spinal cords suggested that potentiation of OPIDN by PMSF was mediated by a mechanism different from that followed by DFP alone to produce OPIDN.     

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

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

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.     

TOCP诱发OPIDN后鸡神经组织MAP-2的变化

目的　探讨微管相关蛋白 2 (MAP -2 )在三邻甲苯磷酸酯 (TOCP)诱发的迟发性神经毒性 (OPIDN)中的含量变化及OPIDN的发病机制。方法　成年罗曼母鸡 18只 ,经口 1次给予TOCP 3 75和 75 0mg kg ,第 2 2天处死动物 ,冷环境下取出大脑、脊髓和坐骨神经 ,匀浆后WesternBlot方法测定MAP 2的含量。结果　TOCP使鸡大脑沉淀中MAP -2在 3 75和 75 0mg/kg组分别升高 2 82 %和 3 60 % ,上清中分别升高 160 %和 2 0 4% ,与对照组相比 ,差异均有显著性 (P <0. 0 1) ;MAP -2在脊髓沉淀中分别降低 5 0 %和 43 % ,脊髓上清中分别降低 2 8%和 5 5 % (P <0 .0 1) ;坐骨神经上清中分别升高 85 %和 3 2 9% (P <0 .0 1) ,坐骨神经沉淀中未检出。结论　TOCP中毒性可引起鸡神经组织中的MAP 2含量发生不同程度改变 ,这种改变可能与TOCP引起的迟发性神经毒性有关。     

Organophosphorus-induced delayed neuropathy: A simple and efficient therapeutic strategy

Organophosphorus (OP) used as pesticides and hydraulic fluids can produce acute poisoning known as OP-induced delayed neuropathy (OPIDN), whose effects take long time to recover. Thus a secure therapeutic strategy to prevent the most serious effects of this poisoning would be welcome. In this study, tri-o-cresyl phosphate (TOCP, 500 mg/kg p.o.) was given to hens, followed or not by nimodipine (1 mg/kg i.m.) and calcium gluconate (Ca-glu 5 mg/kg i.v.). Six hours after TOCP intoxication, neuropathy target esterase (NTE) activity inhibition was observed, peaking after 24 h exceeding 80% inhibition. A fall in the plasmatic calcium levels was noted 12 h after TOCP was given and, in the sciatic nerve, Ca²⁺ fell 56.4% 24 h later; at the same time calcium activated neutral protease (CANP) activity increased 308.7%, an effect that lasted 14 days. Any bird that received therapeutic treatment after TOCP intoxication presented significant signs of OPIDN. These results suggest that NTE may be implicated in the regulation of calcium entrance into cells being responsible for the maintenance of normal function of calcium channels, and that increasing CANP activity is responsible to triggering OPIDN. Thus, with one suitably adjusted dose of nimodipine as well as Ca-glu, we believe that this treatment strategy may be used in humans with acute poisoning by neuropathic OP.     

TRPA1 channel mediates organophosphate-induced delayed neuropathy

OBJECTIVE We want to investigate the mechanism of organophosphate-induced delayed neuropathy(OPIDN) and find appropriate therapeutic medicine.OPIDN,often leads to paresthesias,ataxia and paralysis,occurs in the late-stage of acute poisoning or after repeated exposures to organophosphate(OP) insecticides or nerve agents,and may contribute to the Gulf War Syndrome.METHODS FDSS Ca2~(+)-influx assays,single-cell calcium imaging and patch-clamp electrophysiology were the major testing techniques.Transfected HEK293 cells and dorsal root ganglion(DRG) neurons were used to evaluate the effects of compounds.Wild type and trpa1 knockout mice and adult hyline brown hens were used to evaluate the neuropathological damages caused by the OPs.Transmission electron microscopy imaging was used to observe the nerve injuries ultrastructurally.High-throughput screen for TRPA1 inhibitors was accomplished by Ion Works Barracuda(IWB) automated electrophysiology assay.RESULTS TRPA1(Transient receptor potential cation channel,member A1) channel mediates OPIDN.A variety of OPs,exemplified by malathion,activates TRPA1 but not other neuronal TRP channels.Malathion increases the intracellular calcium levels and upregulates the excitability of mouse DRG neurons in vitro.Mice with repeated exposures to malathion also develop local tissue nerve injuries and pain-related behaviors,which resembles the early symptoms of OPIDN.Both the neuropathological changes and the nocifensive behaviors can be attenuated by treatment of TRPA1 antagonist HC030031 or abolished by knockout of Trpa1 gene.In the classic hens OPIDN model,malathion causes nerve injuries and ataxia to a similar level as the positive inducer tri-ortho-cresyl phosphate(TOCP),which also activates TRPA1 channel.Treatment with HC030031 reduces the damages caused by malathion or TOCP.Duloxetine and Ketotifen,two commercially available drugs exhibiting TRPA1 inhibitory activity,show neuroprotective effects against OPIDN and might be used in emergency situations.CONCLUSION TRPA1 is the major mediator of OPIDN and targeting TRPA1 is an effective way for the treatment of OPIDN.     

Comparative in vitro study of the inhibition of human and hen esterases by methamidophos enantiomers

The current Organisation for Economic Co-operation and Development (OECD) guidelines for evaluating organophosphorus-induced delayed neuropathy (OPIDN) require the observation of dosed animals over several days and the sacrifice of 48 hens. Adhering to these protocols in tests with enantiomers is difficult because large quantities of the compound are needed and many animals must be utilized. Thus, developing an in vitro screening protocol to evaluate chiral organophosphorus pesticides (OPs) that can induce delayed neuropathy is important. This work aimed to evaluate, in blood and brain samples from hens, human blood, and human cell culture samples, the potential of the enantiomeric forms of methamidophos to induce acetylcholinesterase (AChE) inhibition and/or delayed neurotoxicity. Calpain activation was also evaluated in the hen brain and SH-SY5Y human neuroblastoma cells. The ratio between the inhibition of neuropathy target esterase (NTE) and AChE activities by the methamidophos enantiomers was evaluated as a possible indicator of the enantiomers' abilities to induce OPIDN. The (-)-methamidophos exhibited an IC(50) value approximately 6 times greater than that of the (+)-methamidophos for the lymphocyte NTE (LNTE) of hens, and (+)-methamidophos exhibited an IC(50) value approximately 7 times larger than that of the (-)-methamidophos for the hen brain AChE. The IC(50) values were 7 times higher for the human erythrocyte AChE and 5 times higher for AChE in the SH-SY5Y human neuroblastoma cells. Considering the esterases inhibition and calpain results, (+)-methamidophos would be expected to have a greater ability to induce OPIDN than the (-)-methamidophos in humans and in hens.