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.     

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.     

Changes in beclin-1 and micro-calpain expression in tri-ortho-cresyl phosphate-induced delayed neuropathy

Tri-ortho-cresyl phosphate (TOCP) can cause toxic neuropathy known as organophosphate-induced delayed neuropathy (OPIDN), which is pathologically characterized by the swollen axon containing aggregations of neurofilaments, microtubules, and multivesicular vesicles. Autophagy is a self-degradative process which plays a housekeeping role in removing misfolded proteins and damaged organelles. The current study was designed to investigate the possible roles of autophagy in the pathogenesis of OPIDN. Adult hens were treated with a dose of 750mg/kg TOCP by gavage, or injected subcutaneously with 60mg/kg phenylmethanesulfonyl fluoride (PMSF) dissolved in DMSO 24h earlier and subsequently treated with TOCP, then sacrificed on the time-points of 0, 1, 5, 10, and 21 days after dosing of TOCP respectively. The levels of beclin-1 and μ-calpain in tibial nerves and spinal cords were determined by immunoblotting. The results showed that in both tissues TOCP increased the expression of μ-calpain while decreased that of beclin-1. When given before TOCP administration, PMSF pretreatment could protect hens against the delayed neuropathy. In the meantime, pretreatment with PMSF reduced calpain expression below basal and increased beclin-1 expression above basal in tibial nerve, whereas it simply returned calpain and beclin-1 expression to their basal levels in spinal cord. In conclusion, the intoxication of TOCP was associated with a significant change of beclin-1 in hen nervous tissues, which suggested that disruption of autophagy-regulated machinery in neurons might be involved in the pathogenesis of OPIDN.     

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.     

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.     

Delayed neurotoxicity of O-ethyl O-4-nitrophenyl phenylphosphonothioate: Subchronic (90 days) oral administration in hens

Delayed neurotoxicity in hens was produced following daily oral administration of 0.1, 0.5, 1.0, 2.5, 5.0, and 10.0 mg/kg of technical (85%) O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) in gelatin capsules for 90 days. Daily, three groups of hens were given empty gelatin capsules, 10 mg/kg of tri-o-cresyl phosphate (TOCP), or 1 mg/kg of parathion (O,O-diethyl O-4-nitrophenyl phenylphosphorothioate) and served as gelatin capsule controls, positive controls, and negative controls, respectively. TOCP-Treated hens developed delayed neurotoxicity, and those given parathion showed leg weakness with subsequent recovery when the administration of this agent had stopped. The clinical condition of most ataxic hens deteriorated during the 30-day observation period following the end of the oral administration of EPN. Severity of the clinical condition depended on the size of the daily ingested dose, i.e., while hens given small doses showed only ataxia, those treated with large doses progressed to paralysis and died. Days of administration and “total administered dose” before onset of ataxia depended on the daily dose. Degeneration of myelin and axons in the spinal cord was the most consistent histologic change and was identical to that found in TOCP control hens. Only one hen showed sciatic nerve degeneration. Livers from two hens given the highest dose of EPN manifested a moderate degree of hemosiderosis. Plasma cholin esterase was significantly inhibited in all surviving hens given EPN or TOCP at the end of the observation period. A group of hens treated daily with 0.01 mg/kg of EPN showed no abnormality in gait or behavior, and its plasma cholinesterase activity was not significantly different from that of the control. Hens treated with parathion had plasma cholinesterase activity comparable to that of the control 30 days after the administration of the last dose.     

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.     

Delayed neurotoxicity of subchronic oral administration of leptophos to hens: recovery during four months after exposure.

Daily oral administration of small doses of technical grade O-methyl O-4-bromo-2,5-dichlorophenyl phenylphosphonothioate (leptophos, 0.5-20.0 mg/kg) caused delayed neurotoxicity in hens. Severity of clinical condition and progression or improvement of signs of delayed neurotoxicity depended on the dose and duration of administration. Hens given 20.0 mg/kg suffered ataxia, paralysis, and death. Intermediate doses (5 and 10 mg/kg) caused ataxia, with most treated hens showing no change in clinical condition during the 4-mo observation period. Hens given small doses (2.5 and 1.0 mg/kg) demonstrated regression of neurological deficits after administration of leptophos was stopped. Hens given the smallest tested dose (,.5 mg/kg) developed mild ataxia and showed total recovery during the observation period. Days of administration and total administered dose before onset of ataxia depended on the daily dose. Degeneration of axons and myelin i, the spinal cord was the most consistent histopathologic change and was identical to that observed in tri-o-cresyl phosphate (TOCP) control hens. Only one hen, which died early in the treatment period, showed peripheral nerve degeneration. Controls consisted of 3 groups of hens given a daily oral dose of 10.0 mg/kg TOCP, 1.0 mg/kg O,O-diethyl O-4-nitrophenyl phosphorothioate (parathion), or an empty gelatin capsule. TOCP-treated hens developed delayed neurotoxicity, whereas those given parathion showed initial leg weakness but subsequently recovery without developing delayed neurotoxicity. Controls given gelatin capsules remained normal.     

The effects of drug metabolism inducers on the delayed neurotoxicity and disposition of tri-o-cresyl phosphate in hens following a single intravenous administration

Intravenous (iv) administration of tri-o-cresyl phosphate (TOCP) caused a delayed neurotoxic effect in hens similar to that produced by oral and dermal administration. The iv ED50s for producing ataxia and paralysis were estimated to be 15.9 mg/kg and 31.7 mg/kg respectively. The tissue disposition of unaltered TOCP was determined in hens following a single iv injection of 40 mg/kg of TOCP. One hour (hr) after the injection, the leg muscle contained the highest concentration, 26.99 micrograms/g fresh weight followed by the adipose tissue. Among the nerve tissues, the sciatic nerve had the highest concentration, 9.63 micrograms/g followed by the spinal cord and the brain. Except the adipose tissue and the sciatic nerve, the concentration in all analyzed tissues dropped below 1.0 microgram/g (ml) after 24 hr. An unidentified metabolite appeared in bile taken 1 and 3 hr after the injection. Pretreatment of hens with 3-methylcolathrene (3-MC) and beta-naphthoflavone (B-NF) protected against the TOCP-induced delayed neurotoxicity, whereas phenobarbital (PB) failed to protect against the neurotoxicity. Plasma creatine phosphokinase (CK) activity in paralytic birds increased approximately 4 times of the control or symptomless hens on the 21st day. 3-MC-, B-NF- and PB-treatment depressed substantially the concentration of unaltered TOCP in brain and plasma 1 hr after iv dosing with 40 mg/kg of TOCP. Only B-NF pretreatment lowered the level of TOCP in spinal cord. There was no effect of these inducers on the level of TOCP in sciatic nerve and adipose tissue. B-NF and 3-MC lowered significantly the TOCP level in leg muscle, whereas PB had no such effect. More attention should be paid to the role of TOCP in muscle, especially to the leg muscle, judging from the present toxicological and metabolic studies.     

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.     

Delayed neurotoxicity of O-ethyl O-2,4-dichlorophenyl phenylphosphonothioate: effects of a single oral dose on hens.

Delayed neurotoxicity was produced in hens after the administration of a single oral dose of technical (96.43%) O-ethyl O-2,4-dichlorophenyl phenylphosphonothioate (S-Seven) in a gelatin capsule. Doses ranged from 800 to 5000 mg/kg. Hens given 5000 mg/kg also received atropine sulfate to protect them against acute toxicity. S-Seven caused loss of appetite and weight and resulted in ataxia which progressed in some hens to paralysis and death. Controls consisted of four groups of hens given a single oral dose of 500 mg/kg tri-o-cresyl phosphate (TOCP), 10 mg/kg parathion, 30 mg/kg atropine sulfate, or an empty gelatin capsule. TOCP-Treated hens developed delayed neurotoxicity while those given parathion showed weakness but subsequently recovered. Atropine sulfate and gelatin capsule controls remained normal. Degeneration of axons and myelin in the spinal cord was the most consistent histologic change and was identical to that found in TOCP control hens. Only one hen showed sciatic nerve degeneration. Three groups of hens which were given single oral doses of 100, 200, or 400 mg/kg S-Seven showed no neurologic signs of delayed neurotoxicity and their nerve tissues were not damaged. These results add strong confirmative evidence to the hypothesis that delayed neurotoxicity is a general feature of phenylphosphonothioate insecticides.     

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.     

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.     

Brain acetylcholinesterase, acid phosphatase, and 2',3'-cyclic nucleotide-3'-phosphohydrolase and plasma butyrylcholinesterase activities in hens treated with a single dermal neurotoxic dose of S,S,S-tri-n-butyl phosphorotrithioate

The changes in brain acetylcholinesterase (AChE), acid phosphatase (APase), and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP), and plasma butyrylcholinesterase (BuChE) activities were investigated in hens treated with a single, dermal dose (100-1000 mg/kg) of S,S,S-tri-n-butyl phosphorotrithioate (DEF). Three control groups consisted of hens left untreated, given a single, dermal dose of 500 mg/kg tri-o-cresyl phosphate (TOCP, positive control for organophophorous compound-induced delayed neurotoxicity), or 10 mg/kg O,O-diethyl O-4-nitrophenyl phosphorothioate (parathion, negative control). Brain AChE activity, determined 28 days after application, was significantly inhibited in hens given 500-1,000 mg/kg DEF and in TOCP- and parathion-treated hens. In contrast, brain APase and CNP activities were significantly higher in all treatments as compared with those of the untreated hens. Parathion, however, caused the least increase in these enzymatic activities as compared to DEF or TOCP. A single, dermal dose of DEF or TOCP also caused an initial decrease in plasma BuChE activity with maximum depression of enzymatic activity observed 1 to 7 days after administration. This decrease was dose dependent and the enzymatic activity showed partial recovery with time. Hens treated with single, dermal doses of DEF, ranging from 250 to 1000 mg/kg, developed ataxia which progressed to paralysis in some hens. Histopathologic examination revealed axon and myelin degeneration of the spinal cord and peripheral nerves of some hens. The severity and frequency of the neuropathologic lesions were dose dependent. Neurologic dysfunctions and neuropathologic lesions seen in DEF-treated hens were similar to those exhibited in TOCP-treated hens. While parathion produced acute cholinergic effects, it did not cause delayed neurotoxicity. The changes in brain and plasma enzymes are discussed in relation to their role in the pathogenesis of DEF-induced delayed neurotoxicity.     

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。     

Percutaneous toxicity and delayed neurotoxicity of organophosphates in the scaleless hen

The acute toxicity of tri-ortho-cresyl phosphate (TOCP) and the development of delayed neurotoxicity were characterized in the scaleless hen, a featherless mutant, and compared to the responses observed in normally feathered birds. Brain acetylcholinesterase (AChE) activity was comparable between scaleless and normal hens, but nonspecific cholinesterase (ChE) activities of brain and plasma were significantly higher in scaleless birds. The acute ID50 of TOCP for plasma ChE activity was 690 mg/kg for scaleless birds and 240 mg/kg for normal ones following sc administration. However, there was no difference in the ID50 for plasma ChE activity between normal and scaleless hens treated sc with the active metabolite of TOCP, 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one, or parathion. The onset of clinical signs of delayed neurotoxicity in scaleless birds was 8 to 14 days after sc or dermal treatment with TOCP and caused typical axonal fragmentation in the sciatic nerve. Plasma creatine phosphokinase activity was significantly increased following the onset of delayed neurotoxicity in both lines of birds. Dermal application of TOCP to a 50-cm2 area on the backs of scaleless hens inhibited plasma ChE activity in a dose-related manner (ID50 = 115 mg/kg), and the lowest dose of TOCP, 114 mg/kg, did not produce delayed neurotoxicity. The results show that the scaleless hen can be used to determine a no-observable effect level for delayed neurotoxicity which regulatory agencies could use to extrapolate a safe level of human dermal exposure to organophosphates that produce delayed neurotoxicity.     

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.     

Delayed neurotoxicity of O-ethyl O-4-cyanophenyl phenylphosphonothioate (cyanofenphos) in hens

Oral administration of a single dose of cyanofenphos (O-ethyl O-4-cyano-phenyl phenylphosphonothioate) between 10 and 500 mg/kg, caused delayed neurotoxocity in hens similar to that reported for other organophosphorus esters. The severity of the clinical condition depended upon the size of the administered dose. Hens given small doses showed only ataxia, while some of those treated with large doses developed paralysis and died. Hens given 500 mg/kg oral dose of tri-o-cresyl phosphate (TOCP) became paralysed, while those given parathion showed early weakness but subsequently recovered without developing delayed neurotoxicity. Control hens receiving atropine sulfate or gelatin capsules remained normal.     

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     

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.