鲫鱼脑AChE制备及对几种有机磷农药敏感性研究

乙酰胆碱酯酶(AChE)是生物神经兴奋传导中的一种关键性酶。有机磷农药能够磷酰化AChE活性中心的丝氨酸羟基,导致神经传导阻断,因此AChE是有机磷农药残留检测的靶酶。文章选择鲫鱼脑作为酶源,对AChE提取方法进行了研究,并探讨了其对有机磷农药的敏感性。首先粗提AChE,通过正交试验确定了AChE酶活测定的最佳条件,研究了粗酶对几种有机磷农药的敏感性及检测限,之后通过层析方法纯化AChE,得到高活性的目标酶。研究表明AChE活性测定最佳条件为:200mmol/L底物、pH8.0、30℃水浴30min,这些因素对酶得活性影响顺序为:底物浓度pH温度时间,酶剂量对其影响最小;由鲫鱼脑粗提得到的AChE对4种有机磷农药的敏感性为:敌敌畏敌百虫氧乐果三唑磷辛硫磷乙酰甲胺磷;纯化后酶活达到了458.43μmo(lmin·mg),适合用于有机磷农药检测。     

有机磷农药对乙酰胆碱酯酶的抑制作用及QSAR研究

以碘化硫代乙酰胆碱为底物,二硫代二硝基苯甲酸为显色剂,采用分光光度法测定了21种有机磷农药对乙酰胆碱酯酶(AChE)的半抑制浓度(IC₅₀).运用多元线性回归分析以及参数间的自相关分析建立有机磷农药对AChE抑制作用的QSAR模型.模型分析表明,有机磷农药对AChE的抑制作用主要受特定方向上的原子极化率及原子的电拓扑状态的影响,是多因素综合作用的结果.模型可解释高达78%的数据变异和显著的预测能力(q²=0.653),表明有机磷农药对AChE的抑制作用可借助结构活性关系模型进行预测与评价.     

Biochemical interaction of six op delayed neurotoxicants with several neurotargets

Abstract

Five organophosphorous insecticides: Leptophos, EPN, Cyano‐fenphos, trichloronate and salithion proved to cause irreversible ataxia not only to chicken but also to mice and sheep. TOCP was included as a reference. Cyanofenphos blocked the catecholamine B‐receptor binding activity with ³H‐norepinephrine at a level similar to that of the specific inhibitor propranolol in the mouse heart preparation. In the lamb heart preparation, the B‐receptor was more sensitive to Leptophos, salithion and TOCP than to propranolol. The six compounds and their oxons were screened for their in‐vitro inhibition to monamine oxidase (MAO), acetyl cholinesterase (AChE) and neurotoxic esterase (NTE) in the brain of either mouse, lamb or chicken. It is believed that their AChE inhibition stands for their acute toxicity, while NTE inhibition is responsible for their paralytic ataxia.     

太阳鱼脑乙酰胆碱酯酶的分离纯化

采用硫酸铵沉淀–普鲁卡因胺亲和柱层析方法,对太阳鱼(Lepomis gibbosus)脑部乙酰胆碱酯酶分离纯化,纯化倍数为234倍,回收率为25.67%,经SDS-PAGE电泳检测,呈现单一的条带,相对分子质量约为68 000,表明其纯度达到电泳纯.     

Acute toxicity evaluation of triazophos,deltamethrin and their combination on earthworm,Eudrilus eugeniae and its impact on AChE activity

The acute toxicity of three formula grade pesticides namely, triazophos (an organophosphate, OP), deltamethrin (a pyrethroid) and combined pesticide (triazophos?+?deltamethrin) was determined in earthworm, Eudrilus eugeniae. They were exposed to different concentrations of these pesticides for 48 h by paper contact toxicity method. The LC₅₀ values for triazophos, deltamethrin and of combination were determined as 0.076, 0.031 and 0.065?μg/cm², respectively. To study the sublethal effect of these pesticides, E. eugeniae were exposed to 5% and 10% of LC₅₀ of pesticides for 48 h. Morphological alterations such as coiling, clitellar swelling, mucus release and bleeding followed by body segmentation were observed in exposed earthworm. Acetylcholinesterase (AChE) activity assayed in different regions of body segment exhibited a significant (p?<?0.05) decrease in its activity particularly in the pre-clitellar region as compared to other regions. The altered behavioural responses in pesticides exposed earthworms would have been due to decline in AChE activity of the nervous system.     

敌百虫对鲤鱼组织乙酰胆碱酯酶和腺三磷酶活力的影响

在系列浓度的敌百虫农药暴露中,鲤鱼脑乙酰胆碱酯酶(Acetyl-cholinesterase,AChE)和鳃?肾腺三磷酶(Adenosinetriphosphatase,ATPase)的敏感性依次为脑AChE>肾ATPase>鳃ATPase?引起死亡的脑AChE抑制率在急性暴露中可大于95%,而鳃?肾ATPase抑制率达50%~60%时,鱼通常难以存活?实验结果表明,AChE抑制并非是有机磷农药中毒中引起死亡的唯一原因,非胆碱能毒性作用也具有重要的作用?      

二嗪磷对鸟类脑组织乙酰胆碱酯酶活性的影响

为了初步研究有机磷农药二嗪磷对鸟类的毒性作用,采用室内饲喂染毒的方法,分析了二嗪磷对鹌鹑、鹧鸪及斑鸠3种鸟脑组织乙酰胆碱酯酶(AChE)活性的影响。鹌鹑、鹧鸪采用经食饲喂,染毒剂量分别为0、40、80、160、320、600mg·kg-1饲料和0、400、800、1600、3200、6000mg·kg-1饲料,试验周期8d;斑鸠采用拌种饲喂(1mL50%二嗪磷乳油拌500g麦种),染毒剂量为0、1.2、2.0、4.0、8.0mg·kg-1体重,一次性染毒后进行常规饲养,试验周期15d。结果表明,二嗪磷对3种鸟脑组织AChE活性均有一定程度的抑制作用:各染毒组死亡鸟AChE活性较对照降低显著(p<0.05,p<0.01),活性抑制率均超过40%;各染毒组存活鸟AChE活性较对照也有所降低,但活性抑制率均未超过20%.以上结果提示,抑制脑组织AChE活性可能是二嗪磷致鸟类死亡的重要原因。     

Effect of clomazone herbicide on biochemical and histological aspects of silver catfish (Rhamdia quelen) and recovery pattern

The effects of the herbicide, clomazone, on acetylcholinesterase (AChE), catalase and TBARS formation in teleost fish (Rhamdia quelen) were studied. The fish were exposed to 0.5 or 1.0 mg L⁻¹ of clomazone for 12, 24, 48, 96 and 192 h. After 192 h of exposure period, fish were transferred to clean water and kept in the same for 192 h to study the recovery response. Same parameters as that of exposure period were assayed after 96 and 192 h of recovery period. Specific AChE activity was reduced in the brain and muscle after treatments, reaching a maximum inhibition of 47% in the brain and 45% in the muscle after 12 h of exposure. Fish exposed to clomazone increased TBARS production in the liver for all exposure periods. The brain presented elevated TBARS levels after 12, 24 and 48 h, but after 96 and 192 h, these levels decreased. The decrease of TBARS levels persisted in brain tissue after 96 h of recovery and returned to the control value after 192 h in clean water. Catalase activity was reduced for all periods of exposure. Histological analysis showed vacuolation in the liver after herbicide exposure. Some of the alterations observed were completely restored after recovery period.     

久效磷对秀丽隐杆线虫运动、学习和觅食行为的影响

久效磷(MCP)是一种广谱类内吸附性有机磷农药和潜在的神经毒剂,能够对非靶生物产生神经毒性作用,而行为毒性是神经毒性的主要表现形式. 以运动、学习与觅食行为、乙酰胆碱酯酶活性为指标,研究了久效磷对非靶生物——秀丽隐杆线虫(Caenorhabditis elegans,下称线虫)行为的影响. 结果显示:经0.5、5.0和50.0 mg/L久效磷暴露后,各暴露组线虫头部摆动和身体弯曲频率均极显著降低,0.5 mg/L暴露组线虫向前摆动频率显著减少,5.0和50.0 mg/L暴露组线虫向前摆动频率极显著减少;5.0和50.0 mg/L暴露组线虫在NaCl存在和缺少食物条件下,对NaCl的趋向性极显著升高;与对照组相比,50.0 mg/L暴露组线虫菌落接触率在2、4、6、8 h时均显著降低,在24 h时极显著降低;各暴露组线虫的乙酰胆碱酯酶活性均受到极显著抑制. 表明久效磷能够导致线虫运动、学习与觅食行为的严重缺陷,乙酰胆碱酯酶活性抑制与久效磷对线虫运动行为的影响呈正相关,与其学习行为的影响呈负相关.      

Dioxin induces expression of hsa-miR-146b-5p in human neuroblastoma cells

Dioxin can cause a series of neural toxicological effects. Micro RNAs(mi Rs) play important roles in regulating nervous system function and mediating cellular responses to environmental pollutants, such as dioxin. Hsa-mi R-146 b-5 p appears to be involved in neurodegenerative diseases and brain tumors. However, little is known about effects of dioxin on the expression of hsa-mi R-146 b-5 p. We found that the hsa-mi R-146 b-5 p expression and its promoter activity were significantly increased in dioxin treated SK-N-SH cells, a human-derived neuroblastoma cell line. Potential roles of hsa-mi R-146 b-5 p in mediating neural toxicological effects of dioxin may be due to the regulation of certain target genes. We further confirmed that hsa-mi R-146 b-5 p significantly suppressed acetylcholinesterase(ACh E) activity and targeted the3′-untranslated region of the ACh E T subunit, which has been down-regulated in dioxin treated SK-N-SH cells. Functional bioinformatic analysis showed that the known and predicted target genes of hsa-mi R-146 b-5 p were involved in some brain functions or cyto-toxicities related to known dioxin effects, including synapse transmission, in which ACh E may serve as a responsive gene for mediating the effect.