三邻甲苯磷酸酯暴露对鸡脊髓组织基因谱的影响

目的从三邻甲苯磷酸酯(tri-orhto-cresyl phosphate,TOCP)暴露鸡的脊髓组织中筛选有机磷致迟发性神经病(organophosphorus ester induced delayed neuropathy,OPIDN)的相关基因,为进一步探讨OPIDN的发病机制提供目的基因谱。方法罗曼鹤母鸡(Roman hens)随机分为TOCP组、先给苯甲基磺酰氟(phenylmethanesul fonyl fluoride,PMSF)再给TOCP的PMSF前干预组、生理盐水对照组,每组4只。染毒5 d处死实验鸡,低温取脊髓,依次提取总RNA、纯化、转录合成cRNA探针、标记生物素、基因芯片杂交、扫描杂交信号、最后进行基因芯片处理和分析。结果 TOCP组与对照组相比有显著差异,并与PMSF前干预组无显著差异的基因有520个,其中表达差异在3倍以上基因有36个。结论从TOCP暴露鸡脊髓组织中筛选出520个OPIDN相关基因,其中36个基因可能为OPIDN密切相关基因。     

磷酸三邻甲苯酯对母鸡中枢神经组织线粒体中ATP含量及ATPase的影响

目的观察磷酸三邻甲苯酯(tri-ortho-cresyl phosphate,TOCP)对母鸡中枢神经组织线粒体ATP含量及ATP合成酶(ATP synthase,ATPase)的影响,探讨有机磷化合物诱导迟发性神经毒性(organophosphorus ester-induceddelayed neurotoxicity,OPIDN)的机制。方法成年罗曼母鸡96只随机分为实验组和对照组。实验组分别按185、375和750 mg/kg经灌胃一次性染毒TOCP,对照组给予等体积玉米油。分别于染毒1、5、15和21 d处死动物,迅速剥离大脑和脊髓,分离提纯其线粒体,生物发光法检测线粒体ATP含量的改变,并分别采用RT-PCR和Western blotting技术检测了ATPase mRNA水平及蛋白水平的改变。结果实验组与对照组相比,母鸡大脑中线粒体ATP含量没有明显改变,脊髓中3个剂量组均从第5天开始即出现明显降低,第21天线粒体ATP含量较第15天有所升高。RT-PCR结果表明,TOCP染毒后母鸡中枢神经组织中ATPase 6 mRNA水平与正常对照组相比均明显降低。Western blotting结果显示大脑和脊髓中均呈现先升高后降低的趋势,脊髓ATPaseβ蛋白含量变化比大脑变化更明显。结论 TOCP能使母鸡中枢神经组织线粒体ATP含量发生改变,并能引起ATPase mRNA水平和蛋白水平的改变。     

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。     

螺内酯阻抑百草枯中毒大鼠肺纤维化的机制探讨

目的观察螺内酯能否阻抑百草枯（PQ）中毒大鼠肺纤维化及探讨其可能的作用机制。方法54只雄性SD大鼠随机分为对照组、PQ染毒组（一次性PQ80mg／kg灌胃）和螺内酯干预组（一次性PQ80mg／kg灌胃并予每天1次螺内酯100mg／kg灌胃）。各组大鼠于染毒后第7、14、28天分批处死,取肺组织行HE染色、Masson染色并做肺泡炎、肺纤维化评分,计算肺系数,测血浆及肺组织醛固酮（ALD）含量及肺羟脯氨酸含量,应用免疫组化法测肺组织转化生长因子-β1（TGF-β1）及α-平滑肌肌动蛋白（α—SMA）蛋白的表达。结果染毒组和干预组血浆、肺组织ALD含量均较对照组升高,第14天始差异有统计学意义（P〈0．05）;干预组各时间点大鼠肺泡炎和肺纤维化程度积分、肺系数、肺羟脯氨酸含量均低于染毒组（P〈0．01）;干预组第14天始肺组织TGF—β1、α-SMA蛋白的表达均低于染毒组（P〈0．05或P〈0．01）。结论ALD、TGFβ1、α—SMA可能均参与PQ中毒致肺纤维化的发病进程。螺内酯对PQ中毒大鼠肺纤维化有一定程度的阻抑作用,可能与其抑制ALD受体,并减少肺部TGF-β1和aSMA的表达有关。     

百草枯中毒大鼠肺组织ACE/ACE2表达变化及贝那普利的干预作用

目的观察贝那普利对百草枯(PQ)中毒大鼠肺组织病理改变、血管紧张素转化酶(ACE)及其同源物ACE2表达的影响。方法一次性PQ 60mg.kg-1灌胃建立大鼠百草枯中毒肺纤维化模型,干预组染毒后加予贝那普利10mg/(kg.d)灌胃。于染毒后第三、七、二十一天取肺组织行HE染色和Masson染色,观察大鼠肺泡炎、肺纤维化程度;免疫组化测Ⅰ、Ⅲ型胶原;Quantitative real-time PCR检测ACE、ACE2 mRNA表达;ELISA检测肺组织AngⅡ蛋白表达。结果干预组在染毒后3d、7d肺泡炎症减轻;染毒7d后,纤维化程度积分、胶原Ⅰ、胶原Ⅲ含量低于模型组。模型组染毒后各时间点大鼠肺组织ACE mRNA及AngⅡ蛋白表达逐步上调,持续至21d;第七天后ACE2 mRNA的表达降低。干预组各时间点ACE mRNA及AngⅡ蛋白低于模型组,第七天后ACE2 mRNA表达上调。结论贝那普利可能通过降低局部ACE表达或促进ACE2合成,抑制AngⅡ生成,从而减轻百草枯中毒所致肺纤维化形成。     

阻断肾素-血管紧张素-醛固酮系统不同位点对百草枯中毒大鼠肺纤维化的影响

目的 观察通过药物阻断肾素-血管紧张素-醛固酮系统的不同位点对百草桔中毒大鼠肺纤维化的影响及探讨可能的作用机制.方法 SD大鼠一次性百草枯80mg/kg灌胃建立肺纤维化模型,分别每日胃管内灌注生理盐水(染毒组)、卡托普利60 mg/kg、氯沙坦10mg/kg和螺内酯100 mg/kg进行干预.对照组以生理盐水灌胃.各组大鼠均于染毒后第7、14、28 d分批处死,取肺组织行苏木素-伊红(HE)与Masson染色并行肺泡炎、肺纤维化评分,测肺羟脯氨酸(Hyp)含量,用免疫组化法和图像半定量分析系统检测肺组织结缔组织生长因子(CTGF)蛋白的表达.结果 各干预组各时间点大鼠肺泡炎和肺纤维化程度积分、肺Hyp含量、肺组织CTGF蛋白的表达均低于染毒组(P＜0.01).3组采用药物干预后,肺Hyp含量比较的差异有统计学意义(P＜0.05).结论 卡托普利、氯沙坦与螺内酯均能减轻百草枯中毒大鼠肺纤维化的程度,可能通过阻断不同的受体抑制CTGF蛋白的表达而实现.螺内酯减轻肺组织胶原沉积的疗效最佳,氯沙坦最差.     

内毒素血症通过下调HNF4α表达加剧肝损伤

目的 探讨内毒素血症下调肝细胞核因子4α（HNF4α）表达介导肝损伤进展的机制。方法 144只BALB/c小鼠采用随机数字表法分组进行以下实验：（1）四氯化碳（CCl₄）诱导小鼠急性肝损伤。对照组和0.5 mL/kg、1.0 mL/kg、2.0 mL/kg CCl₄组。（2）筛选脂多糖（LPS）干预小鼠的剂量。对照组和0.1 mg/kg、0.5 mg/kg、2.5 mg/kg LPS组。（3）LPS干预CCl₄（1.0 mL/kg）诱导急性肝损伤模型小鼠。对照组、CCl₄组、0.1 mg/kg LPS+CCl₄组、0.5 mg/kg LPS+CCl₄组。每组12只。诱导24 h后处死小鼠,采用酶速率法检测血清丙氨酸转氨酶（ALT）,重氮法检测总胆红素（TBil）水平;Western blot法检测肝组织HNF4α、胱天蛋白酶3剪切体（Cleaved caspase-3）蛋白表达;原位末端标记法检测肝细胞凋亡情况。结果 0.5、1.0、2.0 mL/kg CCl₄组的血清ALT、TBil及肝组织HNF4α、Cleaved caspase-3蛋白表达水平高于对照组,且呈剂量依赖性增高。2.5 mg/kg LPS组血清ALT、TBil及肝组织Cleaved caspase-3蛋白表达水平高于对照组和0.1、0.5 mg/kg LPS组,肝组织HNF4α蛋白表达水平低于对照组和0.1、0.5 mg/kg LPS组（P<0.05）。CCl₄组和0.1、0.5 mg/kg LPS+CCl₄组的血清ALT、TBil,肝组织HNF4α、Cleaved caspase-3蛋白表达水平及肝细胞凋亡指数均高于对照组（P<0.05）;0.1、0.5 mg/kg LPS+CCl₄组的血清ALT、TBil,肝组织Cleaved caspase-3蛋白表达水平及肝细胞凋亡指数高于CCl₄组,HNF4α蛋白表达水平低于CCl₄组（P<0.05）。结论 内毒素血症通过下调HNF4α表达增加肝细胞凋亡,可能是其介导肝损伤进展的机制之一。     

马钱子抑制兔脊髓损伤的细胞凋亡作用

目的：探讨马钱子是否通过抑制细胞凋亡来治疗脊髓损伤。方法：采用改良的Allen＇s法建立兔脊髓损伤模型。设立假脊髓损伤组、对照组、炙马钱子40mg组（M40组）及炙马钱子80mg组（M80组）,分别在给药后1d、7d及用蛋白免疫印迹法（Western blot）检测c-fos蛋白、Bax蛋白和Bcl-2蛋白的表达。结果：与对照组相比,M40组及M80组治疗第7天及14天,可不同程度地抑制损伤脊髓中NO、c-fos蛋白、Bax蛋白的表达,上调Bcl-2蛋白的表达。其中,M40组和M80组分别治疗第7天的NO含量与对照组有统计学差异（P〈0.05）;M80组治疗第7天时c-fos蛋白与对照组有统计学差异（P〈0.05）,治疗14d时有重要统计学差异（P〈0.01）;M80组治疗第7天时Bax蛋白、Bcl-2蛋白及两者的比值与对照组间有统计学差异（P〈0.05）,M80组治疗14d后Bax蛋白与对照组有统计学差异（P〈0.05）。结论：马钱子通过减少损伤脊髓局部NO、c-fos蛋白的表达、下调Bax/Bcl-2比值从而抑制细胞凋亡,阻止脊髓继发性损伤,促进脊髓损伤后神经功能的恢复。     

三邻甲苯基磷酸酯对鸡大脑组织细胞骨架蛋白含量的影响

目的　探讨三邻甲苯基磷酸酯(TOCP)中毒引起迟发性神经毒性的机制。方法　用Western印迹方法测定了TOCP中毒后鸡大脑组织中高分子量神经丝蛋白(NF H)、中分子量神经丝蛋白(NF M)、低分子量神经丝蛋白(NF L)、α微管蛋白、β微管蛋白及β微丝蛋白6种细胞骨架蛋白的相对含量。结果鸡大脑组织沉淀中,NF H在TOCP 3 75和75 0mg·kg- 1 组分别降低2 3 %和4 7%(P <0 .0 1 ) ;而在上清中,分别升高3 1 %(P <0 .0 5 )和2 1 5 %(P <0 .0 1 )。NF M在沉淀中75 0mg·kg- 1 组降低4 7%(P <0 .0 1 ) ;而在上清中分别升高2 4 8%和2 3 3 %(P <0 .0 1 )。NF L在沉淀中75 0mg·kg- 1 组升高1 7%(P <0 .0 5 ) ;在上清中也分别升高2 2 0 %和1 97%(P <0 .0 1 )。α微管蛋白、β微管蛋白和β微丝蛋白在上清和沉淀中相对含量均无明显改变(P >0 .0 5 )。结论　大脑组织中神经丝含量改变较微管和微丝蛋白明显,这种变化可能与TOCP引起的迟发性神经毒性有关     

DNA 6 mA修饰在低剂量镉暴露SD孕鼠脐血中的表达研究

目的通过研究DNA N6-甲基腺嘌呤(N6-methyladenine,6 mA)修饰及其甲基化修饰酶基因在低剂量镉暴露Sprague Dawley(SD)孕鼠脐血中的表达,探讨6 mA表观遗传修饰对镉发育毒性的影响。方法模拟一代繁殖毒性试验构建低剂量镉暴露的SD孕鼠动物模型,以0、1.0、3.0和9.0 mg/kg·bw剂量对SD雌鼠连续灌胃氯化镉溶液90 d后进行交配,查到阴栓者定为孕第0天,持续染毒至孕第20天进行剖杀,收集脐血,提取DNA和RNA,斑点印迹(Dot blot)检测6 mA修饰水平,RT-qPCR检测6 mA修饰酶相关基因的相对表达水平。结果与对照组相比,各染毒组孕鼠之间的各项繁殖指数未见明显的差异,各染毒组胎鼠体重均明显降低(H=47.82,P<0.01),胎鼠身长、尾长等其他发育指标在个别染毒组出现异常。与对照组相比,各染毒组脐血6 mA修饰水平呈下降趋势,但差异无统计学意义(P>0.05);染毒组6 mA甲基化转移酶N6AMT1表达水平较对照组有不同程度下降,其中9.0 mg/kg剂量组N6AMT1表达与对照组相比差异具有统计学意义(P<0.01);去甲基化酶ALKBH1和ALKBH4的表达水平较对照组有不同程度上升,其中3.0和9.0 mg/kg剂量组ALKBH1表达与对照组相比,差异有统计学意义(P<0.05,P<0.01),9.0 mg/kg剂量组ALKBH4表达与对照组相比差异具有统计学意义(P<0.01)。结论SD孕鼠脐血中6 mA修饰酶的基因表达改变与镉暴露相关,DNA 6 mA表观遗传修饰可能对镉的发育毒性有影响。     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.