有机磷农药长期低剂量暴露致认知功能损伤的研究进展

有机磷农药在世界范围内大量使用,其残留农药势必对环境造成持续污染,这种长期、低剂量的有机磷暴露对人类健康的威胁已经引起广泛关注。有机磷致神经毒性主要与胆碱酯酶抑制有关,而流行病学调查显示,环境有机磷暴露人群胆碱酯酶没有明显抑制,但中枢神经系统出现学习记忆等认知功能损伤,其作用机制尚不明确。学习记忆功能的改变不仅受到突触可塑性和乙酰胆碱等神经递质的影响,也与学习记忆相关信号系统的激活,神经细胞骨架的降解以及神经发生等密切相关,随着非胆碱作用机制在有机磷致神经毒性中发挥越来越重要的作用,本文就有长期低剂量机磷农药暴露致认知功能损伤的表现及其可能作用机制的新进展进行简要概述。     

Identification of acylpeptide hydrolase as a sensitive site for reaction with organophosphorus compounds and a potential target for cognitive enhancing drugs

We describe here the purification and identification of a previously unrecognized target for organophosphorus compounds. The target, acylpeptide hydrolase, was isolated as a tritiated-diisopropylfluorophosphate-reactive protein from porcine brain and purified to homogeneity using a combination of ion-exchange and gel-filtration chromatography. Biochemical characterization and internal sequence analysis confirmed identity. Acylpeptide hydrolase was found to be potently inhibited by the organophosphorus compounds chlorpyrifosmethyl oxon, dichlorvos, and diisopropylfluorophosphate (20-min IC(50) values of 18.3 +/- 2.0, 118.7 +/- 9.7, and 22.5 +/- 1.2 nM, respectively). The in vitro sensitivity of acylpeptide hydrolase toward these compounds is between six and ten times greater than that of acetylcholinesterase (AChE), making it a target of pharmacological and toxicological significance. We show that, in vivo, acylpeptide hydrolase is significantly more sensitive than AChE to inhibition by dichlorvos and that the inhibition is more prolonged after a single dose of inhibitor. Furthermore, using dichlorvos as a progressive inhibitor, it was possible to show that acylpeptide hydrolase is the only enzyme in the brain capable of hydrolyzing the substrate N-acetyl-alanyl-p-nitroanilide. A concentration of 154 +/- 27 pmol of acylpeptide hydrolase/gram of fresh rat brain was also deduced by specific labeling with tritiated-diisopropylfluorophosphate. We also suggest that, by comparison of structure-activity relationships, acylpeptide hydrolase may be the target for the cognitive-enhancing effects of certain organophosphorus compounds. Acylpeptide hydrolase cleaves N(alpha)-acylated amino acids from small peptides and may be involved in regulation of neuropeptide turnover, which provides a new and plausible mechanism for its proposed cognitive enhancement effect.     

Computational study of the competitive binding of valproic acid glucuronide and carbapenem antibiotics to acylpeptide hydrolase

The efficacy of the antiepileptic drug VPA is decreased by co-administered carbapenems (CBPMs). The mechanism of CBPM selective inhibition of acylpeptide hydrolase (APEH) hydrolysis of VPA-glucuronide (VPA-G) to VPA is unclear due to the lack of APEH structural information. Here we performed homology modeling of the three-dimensional structure of APEH and subsequent docking simulations with a modeled structure to understand this mechanism. Docking simulations indicated that four groups of binding structures were involved in the binding of VPA-G, panipenem, and meropenem to APEH, but only one or two binding structures were involved in the binding of meropenem with an open β-lactam ring structure and other antibiotics involving ampicillin. One of the four VPA-G binding structures was close enough to the APEH catalytic triad to facilitate VPA-G hydrolysis. This binding structure was also the most stable binding structure for panipenem, suggesting potential inhibition of VPA-G hydrolysis by panipenem. Fragment molecular orbital calculations of interaction energies of amino acid residues of APEH with VPA-G, panipenem, and meropenem indicated that the binding structure for panipenem closest to the catalytic triad is stabilized upon APEH interaction. These data suggest that APEH binding characteristics with CBPMs may help explain the selective inhibition of APEH by CBPMs.     

Noncholinesterase Effects Induced by Organophosphate Pesticides and their Relationship to Cognitive Processes: Implication for the Action of Acylpeptide Hydrolase

Organophosphate pesticides have been classically described as inhibitors of acetylcholinesterase (AChE) activity in insects and invertebrates. However, there is now more evidence supporting the hypothesis that these compounds also act through noncholinergic pathways, especially those related to cognitive processes. The enzyme acylpeptide hydrolase was identified as a new target for organophosphate pesticides. This enzyme is more sensitive than AChE to some organophosphates (OP), including dichlorvos, which is the parent compound for metrifonate, a therapeutic agent used in the treatment of cognitive impairment associated to Alzheimer´s disease. Therefore, there is some doubt as to whether the mechanism of action of this drug is mediated by a potentiation of cholinergic transmission. However, the direct action of acylpeptide hydrolase in cognitive processes and the physiological and molecular mechanisms underlying subacute exposure to OP have yet to be demonstrated. This review deals with evidence demonstrating the existence of mechanisms of actions of OP, which are independent of cholinergic pathway potentiation and which have an effect on cognitive processes. In addition, the possible participation of the enzyme acylpeptide hydrolase in these processes is also discussed. Finally, the possibility of using this enzyme activity as a new biomarker for exposure to OP is considered.     

Stereochemical considerations on the inhibition of hepatic epoxide hydrolase by some pesticides and their epoxides

The present studies identify the steric factors involved in the hydration of epoxide intermediates of some pesticides by hepatic epoxide hydrolase. Investigations were carried out regarding the formation of reactive epoxide intermediates and their different interactions with the hepatic epoxide hydrolase. Some pesticides and their parent epoxides were selected on he basis of the steric hindrance on the oxirane ring. The results indicate that the inhibition of this enzyme depends on the steric hindrance produced by substituents on the oxirane ring of these pesticides. Mono- and di-substituted oxiranes are good substrates of the epoxide hydrolase and non-competitive inhibitors of the hydration of styrene oxide, while tri-substituted epoxides are virtually inactive on inhibiting the hepatic epoxide hydrolase activity.     

The role of multifunctional drug therapy as an antidote to combat experimental subacute neurotoxicity induced by organophosphate pesticides

Organophosphate pesticides are used in agriculture where they are associated with numerous cases of intentional and accidental misuse. These toxicants are potent inhibitors of cholinesterases leading to a massive build‐up of acetylcholine which induces an array of deleterious effects, including convulsions, oxidative damage and neurobehavioral deficits. Antidotal therapies with atropine and oxime yield a remarkable survival rate, but fail to prevent neuronal damage and behavioral problems. It has been indicated that multifunction drug therapy with potassium channel openers, calcium channel antagonists and antioxidants (either single‐agent therapy or combination therapy) may have the potential to prevent cell death and/or slow down the processes of secondary neuronal damage. The aim of the present study, therefore, was to make a relative assessment of the potential effects of nicorandil (2 mg/kg), clinidipine (10 mg/kg), and grape seed proanthocyanidin (GSPE) extract (200 mg/kg) individually against subacute chlorpyrifos induced toxicity. The test drugs were administered to Wistar rats 2 h after exposure to Chlorpyrifos (CPF). Different behavioral studies and biochemical estimation has been carried in the study. The results showed that chronic administration of CPF significantly impaired learning and memory, along with motor coordination, and produced a marked increase in oxidative stress along with significantly reduced acetylcholine esterase (AChE) activity. Treatment with nicorandil, clinidipine and GSPE was shown to significantly improve memory performance, attenuate oxidative damage and enhance AChE activity in rats. The present study also suggests that a combination of nicorandil, clinidipine, and GSPE has a better neuroprotective effect against subacute CPF induced neurotoxicity than if applied individually. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1017–1026, 2016.     

Acute alcohol effects on cognitive function in social drinkers: their relationship to drinking habits

Abstract Rationale. Several studies suggest that cognitive deficits seen in late stages of alcoholism are related to executive function. However, little is known about the acute effects of alcohol on cognitive executive functions. Aims. The present investigation examined the acute effects of a moderate alcohol dose on tests of planning and spatial working memory as well as on tests of spatial and pattern recognition. The relationship between the acute alcohol effects on performance in these tasks and extreme drinking patterns were also studied. Methods. Alcohol (0.8 g/kg) or placebo was administered to 95 social drinkers. In the planning task, alcohol decreased the number of solutions with the minimum moves. Alcohol also decreased the thinking time before initiating a response, while it increased the subsequent thinking time in the same task. Under alcohol, participants recognised fewer items in the spatial recognition task; however no effect of alcohol was found in a spatial working memory task and in a pattern recognition task. Among the participants with moderate to heavy use of alcohol, those who were 'bingers' performed worse in the spatial working memory and in the pattern recognition task than 'non-bingers'; no interaction between treatment and drinking pattern was found. Conclusion. These data suggest that alcohol given acutely impairs executive-type cognitive functions and that binge drinking may be associated with impaired cognitive function in a working memory and a pattern recognition task. Electronic Publication     

In vivo inhibition of acylpeptide hydrolase by carbapenem antibiotics causes the decrease of plasma concentration of valproic acid in dogs

1.?Our previous in vitro studies suggest that inhibition of the acylpeptide hydrolase (APEH) activity as valproic acid glucuronide (VPA-G) hydrolase by carbapenems in human liver cytosol is a key process for clinical drug–drug interaction (DDI) of valproic acid (VPA) with carbapenems. Here, we investigated whether in vivo DDI of VPA with meropenem (MEPM) was caused via inhibition of APEH in dogs.

2.?More rapid decrease of plasma VPA levels and increased urinary excretion of VPA-G were observed after co-administration with MEPM compared with those after without co-administration, whereas the plasma level and bile excretion of VPA-G showed no change.

3.?Dog VPA-G hydrolase activity, inhibited by carbapenems, was mainly located in cytosol from both the liver and kidney. APEH-immunodepleted cytosols lacked VPA-G hydrolase activity. Hepatic and renal APEH activity was negligible even at 24?h after dosing of MEPM to a dog.

4.?In conclusion, DDI of VPA with carbapenems in dogs is caused by long-lasting inhibition of APEH-mediated VPA-G hydrolysis by carbapenems, which could explain the delayed recovery of plasma VPA levels to the therapeutic window even after discontinuation of carbapenems in humans.     

Some further data on the effects of two organophosphate pesticides on the oxidative metabolism in the liver

Sumithion is the most active cholinesterase inhibitor. The cholinesterase inhibiting action of the organophosphates (OPs) is better compensated by vitamin E in normal animals, but by vitamin A in vitamin A-deficient animals. The lipid peroxidation (LP) is enhanced by the antioxidant vitamins in the liver of normal rats; although they decrease it in the liver of vitamin A-deficient animals, in no case do they prevent the LP-enhancing effect of the OPs examined. The OPs examined inhibit protein synthesis in the liver of vitamin A-deficient animals.     

Evaluation of DNA damage and cytotoxicity induced by three commonly used organophosphate pesticides individually and in mixture,in rat tissues

Organophosphate pesticides are among the most widely used synthetic chemicals for controlling a wide variety of pests. Chlorpyrifos (CPF), methyl parathion (MPT), and malathion (MLT) are among the most extensively used organophosphate (OP) pesticides. The main target of action of OP compounds is the central and peripheral nervous system, although it has also been postulated that these compounds in both acute and chronic intoxication, disturb the redox processes and thus induce oxidative stress. The excessive generation of reactive oxygen species (ROS) causes damage to all vital macromolecules including lipids, proteins, and DNA. This study was aimed to investigate the genotoxicity and cytotoxicity of CPF, MPT, and MLT when given singly or in combination. The DNA damage was measured by alkaline single‐cell gel electrophoresis or comet assay and expressed as DNA damage index. The results showed that both acute and chronic exposure with CPF, MPT, and MLT, caused significantly marked DNA damage in rat tissues namely, liver, brain, kidney, and spleen, when measured 24 hour posttreatment. It was also observed that MPT caused highest level of DNA damage and brain was maximally affected by these OP compounds. When these pesticides were given in mixture, the damage was not the sum of damage caused by individual pesticide, confirming that these pesticides do not potentiate the toxicity of each other. When the DNA damage was measured 48 and 72 hour posttreatment, the damage was partially repaired. Pesticide exposure also caused histopathological changes in rat tissues. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 543–552, 2013.     

Estimation of the LD1 and extrapolation of the LD0.1 for five organophosphate pesticides

The oral LD_50,s for 5 Organophosphate pesticides have been determined in CD-1 strain male and female mice. The values in mg/kg are: Triohlorfon, 800 and 800; Naled, 409 and 330; Dichlorvos, 139 and 133, GC6506, 23.4 and 17.8; Fospirate, 225 and 263 respectively. Toxicity was greater in males with Fospirate and greater in females with Naled and GC6506. The predicted LD₁'s and the extrapolated LD_0.1's have been determined for the 5 organophosphates from an unbalanced design, loaded heavily toward the lower end of the dose-response curve. It has been shown that the slopes of the curves obtained with 50, 100 and 660 animals are parallel for all compound except Fospirate in the 660 mouse experiments. This is probably related to excessive female deaths in the upper segment of the dose-response curve. Sex dependent lethality was observed with Trichlorfon, Dichlorvos and Fospirate with the males being more susceptible than the females except in the case of Fospirate where there was a reversal at the LD₅₀ with greater susceptibility in the females. The conditions for obtaining accurate results in such experiments have been established. The implications of human exposure to low levels of the environmental pollutants have been discussed.     

Inhibition of 5-hydroxytryptamine reuptake by amitriptyline and zimelidine and its relationship to their therapeutic action

Rats were rendered tolerant to ethanol by daily gavage of 4–5 g/kg. The degree of motor impairment on the moving belt test and of hypothermia after i.p. test doses of ethanol was measured prior to and at various times during the chronic treatment, to assess the rates of tolerance development. l-Tryptophan (75 mg/kg twice daily) was administered chronically to elevate brain serotonin level. This treatment did not alter the motor impairment or hypothermia produced by the initial test doses of ethanol (2.0 and 2.5 g/kg respectively). However, the development of tolerance to both the motor impairment and hypothermia effects of ethanol was accelerated in the tryptophan-treated rats. This finding complements our earlier observations that depletion of 5-HT with p-CPA slows down tolerance. Blood ethanol measurements at 20 min (motor impairment) or 90 min (hypothermia) after the administration of the test dose reveal no significant difference between the control and tryptophan-treated rats, suggesting that tryptophan did not influence the metabolism of ethanol. This finding supports the hypothesis that brain serotonin modulates the development of tolerance to ethanol.     

Antinociceptive effects induced by desipramine and fluoxetine are dissociated from their antidepressant or anxiolytic action in mice

This study aimed to evaluate the relationship between some aspects of experimental depression, anxiety and the antinociceptive effects of fluoxetine and desipramine in mice. Acute administration of fluoxetine and desipramine (5, 10 and 20 mg/kg, i.p.) showed significant antinociceptive effects in the hot-plate test and against the early and late phase of the mouse formalin test, dissociated from its antidepressant and anxiolytic effects as measured in the forced swimming and in the plus-maze tests, respectively. Neither fluoxetine nor desipramine, at the doses tested, produced significant effects on locomotor activity. Furthermore, both compounds were ineffective in the tail-flick phasic model of nociception. In conclusion, the results suggest that without the distinction of serotonergic and noradrenergic contributions, the acute antinociceptive effects of fluoxetine and desipramine in mice are independent of their sedative, antidepressant and anti-anxiety properties.     

宾赛克嗪对有机磷农药所致循环衰竭的救治作用

目的：评价宾赛克嗪对胆碱酯酶抑制剂敌敌畏和失血所致循环衰竭的救治效果。方法：健康Wistar♂大鼠42只,随机分为6组：敌敌畏染毒后采用宾赛克嗪0.5、1.0、2.0mg/kg救治组;失血性休克后生理盐水救治组及宾赛克嗪0.5、2.5mg/kg救治组,每组7只。敌敌畏染毒组采用累积染毒,直至平均动脉压（MBP）降至45mmHg（1mmHg=0.133kPa）为循环衰竭标准。失血性休克模型组从股动脉快速放血,15min内使MBP降至休克水平（35～40mmHg）,维持此血压水平30～60min即造成失血性休克模型,然后救治。观察休克过程及救治后血流动力学指标及心电图变化。结果：与染毒前相比,敌敌畏所致大鼠循环衰竭时,SBP、DBP、MBP、心率以及反映心脏收缩功能的左室内压上升段最大变化速率（＋dp/dtmax）、心肌纤维缩短速度（Vpm）和＋dp/dtmax/IP（等容收缩期压力）,反映心脏舒张功能的左室内压下降段最大变化速率（-dp/dtmax）、左室舒张压（LVDP）和IP功能均显著降低（P〈0.01）。心电图显示：心率缓慢,有心律失常。敌敌畏导致的大鼠循环衰竭,在宾赛克嗪0.5mg/kg救治后3min、宾赛克嗪1.0mg/kg救治后2min、宾赛克嗪2.0mg/kg救治后1min,各个指标均脱离循环衰竭水平,在观察点60min内血压维持较平稳。心电图基本在救治后3min恢复正常。单纯给予宾赛克嗪对失血性休克大鼠的血压有一定的改善作用。宾赛克嗪0.5mg/kg较2.5mg/kg起效时间慢,但维持时间长,且对SBP及DBP均有改善作用;宾赛克嗪2.5mg/kg起效时间快,但作用时间短暂,对SBP改善作用明显,对DBP改善作用较小。结论：新型抗毒剂宾赛克嗪对有机磷农药引起的循环衰竭治疗效果更好,是一种良好的新型抗毒剂。     

Neuropsychological and psychiatric functioning in sheep farmers exposed to low levels of organophosphate pesticides

The study aim was to determine whether low level exposure to organophosphate pesticides (OPs) causes neuropsychological or psychiatric impairment. Methodological weaknesses of earlier studies were addressed by: recruiting participants who had retired on ill health grounds; excluding participants with a history of acute poisoning, medical or psychiatric conditions that might account for ill health; and exploring factors which may render some individuals more vulnerable to the effects of OPs than others. Performance on tests of cognition and mood of 127 exposed sheep farmers (67 working, 60 retired) was compared with 78 unexposed controls (38 working, 40 retired) and published test norms derived from a cross section of several thousand adults in the general population. Over 40% of the exposed cohort reported clinically significant levels of anxiety and depression compared to less than 23% of controls. Exposed subjects performed significantly worse than controls and standardisation samples on tests of memory, response speed, fine motor control, mental flexibility and strategy making, even after controlling for the effects of mood. The pattern was similar for both working and retired groups. The cognitive deficits identified cannot be attributed to mood disorder, malingering, a history of acute exposure or genetic vulnerability in terms of PON1₁₉₂ polymorphisms. Results suggest a relationship may exist between low level exposure to organophosphates and impaired neurobehavioural functioning and these findings have implications for working practice and for other occupational groups exposed to OPs such as aviation workers and Gulf War veterans.     

有机磷农药诱变性的构效关系分析及分子机理

通过建立整体结构模式化多因素判别分析法(WSPMDA),对有机磷农药三种构份的亚型进行了有代表性的模式化,进而排列组合成36种整体模式,据有机磷农药诱变性测试资料,用该法推算出各整体模式在6种诱变试验中的预期阳性概率P(M~+),并分析每种构份的亚型模式与诱变性关系的密切程度.观察到三种构份均对有机磷的诱变性几乎在每种试验中都有影响,讨论了如何正确应用所得P(M~+)、还就分析结果结合诱变试验原理推测有机磷诱变分子机理存在三条不同途径。     

Poisoning of an urban family due to misapplication of household organophosphate and carbamate pesticides.

A case report of an urban family who experienced excessive exposure to organophosphate and carbamate pesticides is presented. All three family members developed symptoms that were compatible with cholinesterase inhibition: headache, lightheadedness, wheezing, shortness of breath, nausea, and fatigue. Serial measurement of red blood cell and serum cholinesterases soon after exposure and during subsequent months confirmed the diagnosis of pesticide poisoning. This report demonstrates that the misapplication of pesticides commonly used in residences in urban areas can cause acute pesticide poisoning and demonstrates the usefulness of repeated measurements of cholinesterase during the post-exposure period in establishing the correct diagnosis.     

Regulating pesticides in the UK: a case study of risk management problems relating to the organophosphate diazinon.

OBJECTIVES: (1) To assess aspects of occupational and related environmental health risk assessment and risk management decisions of UK regulatory bodies on diazinon used in sheep dip; and (2) to benchmark those decisions against 'the public health precautionary approach'. METHODS: Analysis of diazinon health and safety data available within Government Departments, industry and from users in animal husbandry practice. RESULTS: (1) Data on diazinon produced by the manufacturing companies for the UK pesticide regulatory agencies are not fully transparent; (2) UK regulatory health and safety processes assume accuracy of manufacturer's data and information provided on personal protective equipment (PPE) and application effectiveness; (3) data available reveal gaps and problems with diazinon toxicity, PPE and application methods; and (4) little published evidence shows that industry followed up the health of dippers after product registration or that government departments adopted a public health approach to regulation. CONCLUSIONS: Diazinon sheep dip illustrates the need for the application of a rigorous precautionary principle in both initial registration and later monitoring of chemicals.