Novel protein targets for organophosphorus pesticides in rat brain

We report preliminary results from a proteomic search for rat brain protein targets adducted by organophosphorous pesticides. Azamethaphos, chlorfenvinphos, diazinon, malathion and chlorpyrifos oxons (in rat brain homogenates) or pirimiphos-methyl (after systemic treatment) were tested at levels producing no more than 30% inhibition of brain acetylcholinesterase. Loss of reactivity with tritiated diisopropylflurophosphate was taken as proof of adduction by the test organophosphate. In addition to acetylcholinesterase other, previously unrecognised, adducted proteins were detected in total brain protein extracts at 30, 32, 41, 71 and 83 kDa. Azamethiphos adducted all but the 30 and 32 kDa bands, but chlorpyrifos only acetylcholinesterase.     

Electroretinographic changes induced by organophosphorus pesticides in rats.

Electroretinographic changes induced by organophosphorus pesticides (OPs) were studied in rats. Male Wistar rats were intraperitoneally injected with fenthion, chlorpyrifos, fenitrothion, dichlorvos or chlorfenvinphos at doses of 0.01 mmol/kg and/or 0.05 mmol/kg. The electroretinogram (ERG) was recorded at 5 hours and 2 days after the administration, and brain and retinochoroid cholinesterase (ChE) activities was assayed at 3 days after the injections. The brain and retinochoroid ChE activities were reduced in rats treated with the OPs. Notably, the reduction of ChE activities by fenthion, chlorpyrifos and dichlorvos were similar. The administration of OPs induced a change in the ERG, characterized by alteration of the amplitudes of a- and b-waves. Nevertheless the ChE activities in the brain and retinochoroid were inhibited by all of the OPs, the OPs affected the amplitude of ERG differently. Fenthion and chlorpyrifos decreased the amplitudes; dichlorvos and chlorfenvinphos increased; and fenitrothion transiently decreased at 5 hours but increased 2 days after the injection. These results indicate that a factor or factors other than inhibition of ChE activities contributes to the alteration of ERG induced by OPs.     

Cytogenetic effects induced by organophosphorus pesticides in mouse spermatocytes

Male mice (Q strain) received a single i.p. injection of 14 organophosphorus compounds, including 11 insecticides, administered on separate occasions. After a recovery period of 10 to 15 days, the cytogenic effects were analyzed in primary spermatocytes at diakinesis-metaphase I corresponding to the treatment of A4-B type spermatogonia. At the highest tolerated dose, trimethylphosphate (1000 mg/kg), triethylphosphate (300 mg/kg), dichlorvos (10 mg/kg), methylparathion (10 mg/kg), ethylparathion (10 mg/kg), ethylparaoxon (0.3 mg/kg), fenitrothion (1000 mg/kg), methylbromophos (1000 mg/kg), ethylbromophos (1000 mg/kg), dimethoate (10 mg/kg), malathion (300 mg/kg), methylazinphos (1 mg/kg), ethylazinphos (1 mg/kg) and trichlorfon (100 mg/kg) did not produce chromosome damage.     

Estrogenicity of organophosphorus and pyrethroid pesticides

Although organophosphorus and pyrethroid pesticides are considered environmental contaminants, their estrogenic potentials are still ubiquitous and unclear. The present study was undertaken to evaluate the estrogenic activities of nine pesticides (phoxim, malathion, monocrotophos, dimethoate, opunal, fenvalerate, cypermethrin, permethrin, and deltamethrin) using three in vitro methods [E-Screen assay, estrogen receptor (ER) competitive binding assay, and pS2 expression assay]. All the pyrethroid pesticides tested induced MCF-7 cell proliferation significantly, while organophosphorus pesticides did not. The estrogenic potency were ranked as permethrin > fenvalerate > cypermethrin > deltamethrin. The proliferation induced by cypermethrin, permethrin, and deltamethrin was blocked by ICI 182.780, while fenvalerate only partly inhibited it. In addition, pyrethroid pesticides inhibited the binding of [3H]estradiol to ER, while the organophosphorus failed to do so. Fenvalerate, permethrin, and cypermethrin induced pS2 mRNA expression with varying potency, while there were no significant effects in deltamethrin-treated groups. Our findings provide evidence to support the idea that pyrethroid pesticides tested produce an ER-specific, agonist response. Fenvalerate induced MCF-7 cell proliferation by a mechanism not involving ER-mediated pathway. Organophosphorus pesticides tested showed no estrogenic potential. Compared with the pS2 expression assay, E-Screen was a more sensitive and useful assay for screening of the xenoestrogenic chemicals.     

Fatty acid amide hydrolase inhibition by neurotoxic organophosphorus pesticides

Organophosphorus (OP) compound-induced inhibition of acetylcholinesterase (AChE) and neuropathy target esterase explains the rapid onset and delayed neurotoxic effects, respectively, for OP insecticides and related compounds but apparently not a third or intermediate syndrome with delayed onset and reduced limb mobility. This investigation tests the hypothesis that fatty acid amide hydrolase (FAAH), a modulator of endogenous signaling compounds affecting sleep (oleamide) and analgesia (anandamide), is a sensitive target for OP pesticides with possible secondary neurotoxicity. Chlorpyrifos oxon inhibits 50% of the FAAH activity (IC50 at 15 min, 25 degrees C, pH 9.0) in vitro at 40--56 nM for mouse brain and liver, whereas methyl arachidonyl phosphonofluoridate, ethyl octylphosphonofluoridate (EOPF), oleyl-4H-1,3,2-benzodioxaphosphorin 2-oxide (oleyl-BDPO), and dodecyl-BDPO give IC50s of 0.08--1.1 nM. These BDPOs and EOPF inhibit mouse brain FAAH in vitro with > or =200-fold higher potency than for AChE. Five OP pesticides inhibit 50% of the brain FAAH activity (ED50) at <30 mg/kg 4 h after ip administration to mice; while inhibition by chlorpyrifos, diazinon, and methamidophos occurs near acutely toxic levels, profenofos and tribufos are effective at asymptomatic doses. Two BDPOs (dodecyl and phenyl) and EOPF are potent inhibitors of FAAH in vivo (ED50 0.5--6 mg/kg). FAAH inhibition of > or =76% in brain depresses movement of mice administered anandamide at 30 mg/kg ip, often leading to limb recumbency. Thus, OP pesticides and related inhibitors of FAAH potentiate the cannabinoid activity of anandamide in mice. More generally, OP compound-induced FAAH inhibition and the associated anandamide accumulation may lead to reduced limb mobility as a secondary neurotoxic effect.     

Age-related differences in sensitivity to organophosphorus pesticides

Organophosphorus (OP) pesticides are used extensively throughout the world to control undesirable pest species. The primary mechanism of action for OP insecticides is inhibition of acetylcholinesterase (AChE), an enzyme dynamically involved in cholinergic neurotransmission. Extensive inhibition of AChE leads to accumulation of acetylcholine in the synapse, disruption of normal impulse flow and subsequent signs of toxicity, including autonomic dysfunction, involuntary movements, muscle fasciculations and a host of others. It is generally believed that young individuals are more sensitive to the neurotoxic effects of these agents relative to adults. Essentially all studies addressing age-related differences in sensitivity to these toxicants have examined responses to acute exposures, however, using acute toxicity (lethality) as the endpoint. As the biochemical mechanism of toxicity for this class of toxicants (inhibition of AChE) is well known and considering that low level, repeated exposures are of great concern to the general public, we propose that evidence of neurochemical alterations, especially when exposures occur during development and maturation, is a more relevant endpoint of toxicity than lethality for estimating susceptibility. This report briefly summarizes previous and ongoing work in our laboratory which examines the relative sensitivity to these toxicants between young and adult rats.     

Rapid simultaneous determination for organophosphorus pesticides in human serum by LC-MS

A simple and rapid method was developed for measuring 10 organophosphorus pesticides (acephate, methidathion, dichlorvos, fenthion, EPN, diazinon, phenthoate, malathion, fenitrothion, and cyanophos) in the serum of acute poisoning patients by LC/MS. Following deproteinization by acetonitrile, an aliquot of the biological sample was injected into a C(18) column using 10mM ammonium formate-methanol as the mobile phase. Extraction recoveries were satisfactory and ranged between 60.0 and 108.1% in serum. The limits of detection (LODs) in serum ranged from 0.125 to 1 microg/ml, and the limits of quantitation (LOQs) ranged from 0.25 to 1.25 microg/ml. An excellent linearity was observed for these LOQs up to 8 microg/ml. Intra- and interassay precision and accuracy were satisfactory for most of the pesticides analyzed. In terms of temperature stability, of all the organophosphorus compounds analyzed, dichlorvos and malathion exhibited the most rapid degradations over 24h at room temperature. Methidathion and diazinon remained relatively stable at all temperatures during the entire 4-week testing period. The present method was successfully applied to one actual case of acute poisoning. In conclusion, this method is simple, accurate, and useful for the determination of organophosphorus pesticides and should benefit both clinical and forensic toxicology.     

Differential signatures of protein expression in marmoset liver and thymus induced by single-dose TCDD treatment

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an ubiquitously distributed environmental pollutant. Health effects have been studied intensively, but low-dose effects are quite complex and not yet fully understood. In many studies, the immune system was identified as the most sensitive target. Here, we demonstrate changes of protein expression in liver and thymus of male marmosets (Callithrix jacchus) which were subjected to a single dose of a subcutaneous injection of 100 ng/kg body weight TCDD. Histopathological examination revealed myocardial fibrosis, but there were no significant findings in pathology and histopathology of liver and thymus. In order to detect more subtle treatment-related changes, we performed a comparative proteomic investigation of liver and thymus using a 2-D gel electrophoresis based proteomics approach. Fluorescence labeling and automated image analysis was used to enhance sensitivity and reproducibility. In both organs, distinct changes of protein expression were detected which were more pronounced in thymus, where the pattern of deregulated proteins could be clearly related to immune responses. In the thymus of treated animals, several toxicologically relevant factors were increased, including chaperones, glycerol-3-phosphate dehydrogenase, and adseverin. Among others, vimentin, Ca-dependent protease and protein disulfide isomerase were downregulated. In the liver, transferrins, lamin A and HSP70 were upregulated, whereas thymidine phosphorylase (synonyms: endothelial cell growth factor, PD-ECGF, gliostatin) was significantly reduced. Comparative analysis of deregulated proteins in both organs revealed a pattern of related functions, which fits well into the existing knowledge of the toxic processes and mechanisms underlying TCDD-mediated toxicity.     

24种有机磷农药的诱变性

本文用6种短期诱变试验(SAL,ECO,SUB,MNM,SOS,MNC)测试了24种有机磷农药的诱变性,结果表明不仅甲基有机磷,而且乙基有机磷和一些其他有机磷均可具有诱变性,并且在有些试验中乙基有机磷还可达到较高的阳性率,在不同试验中甲基有机磷和乙基有机磷阳性率的差异是由于试验反映的遗传学终点的差异和试验系统对有机磷生物转化解毒能力的差异所致.通过观察S9对有机磷诱变性的影响,说明有机磷属直接诱变刑.文中还提出检测有机磷诱变性合理的试验组合。     

川白芷药材中有机磷类农药残留量的测定

目的 制定川白芷药材中有机磷类农药最大残留的限量标准(MRLs).方法采用乙酸乙酯冰浴超声提取,SPE活性炭固相萃取柱净化除杂质,GC - NPD法检测药材中有机磷类农药的残留量.结果建议将川白芷药材中有机磷类农药的最大残留限量规定为:敌敌畏0.2μg·g-1、乙酰甲胺磷0.5μg·g-1、乐果0.2 μg·g-1、马...     

Neurotoxic effects in patients poisoned with organophosphorus pesticides

In this paper we review neurotoxic disorders appearing in patients poisoned with organophosphorus pesticides. These compounds cause four important neurotoxic effects in humans: the cholinergic syndrome, the intermediate syndrome, organophosphate-induced delayed polyneuropathy (OPIDP) and chronic organophosphate-induced neuropsychiatric disorder (COPIND). Compared to the cholinergic syndrome, that causes millions of cases of poisoning each year, other disorders involve much smaller numbers of patients. The review is focused on the neurotoxic effects appearing after acute and chronic exposure to organophosphates with emphasis on clinical presentation, pathogenesis, molecular mechanisms, and possibilities for prevention/therapy.     

薄层色谱扫描法和气相色谱/质谱法快速诊断有机磷农药中毒

目的建立薄层色谱法快速定性和气相色谱(GC)/质谱(MS)法定性定量检测有机磷农药,快速诊断有机磷农药中毒。方法检材经二氯甲烷萃取后,薄层色谱法快速定性,GC/MS选择离子模式定性定量检测检材中有机磷农药浓度。结果对硫磷、甲拌磷、马拉硫磷、辛硫磷和久效磷在紫外灯下均有较好的紫外吸收,用双波长薄层色谱扫描仪对斑点做原位紫外吸收扫描,最大吸收波长分别为300,240,225,290,260nm,可配合比移值进行定性分析;GC/MS选择离子检测方式进行定性定量检测,各药物的特征离子峰分别为:97,109,291;76,97,121;93,125,173;77,97,129;67,97,127。结论薄层色谱扫描法定性检测检材中有机磷农药,具有快速、准确、操作简便等特点,加上GC/MS定性定量检测,不仅提高了方法的灵敏度,而且使结果更加准确可靠,可应用于有机磷农药中毒的快速诊断和有机磷农药中毒致死的法医学鉴定。     

Medical treatment of acute poisoning with organophosphorus and carbamate pesticides

Organophosphorus compounds (OPs) are used as pesticides and developed as warfare nerve agents such as tabun, soman, sarin, VX and others. Exposure to even small amounts of an OP can be fatal and death is usually caused by respiratory failure. The mechanism of OP poisoning involves inhibition of acetylcholinesterase (AChE) leading to inactivation of the enzyme which has an important role in neurotransmission. AChE inhibition results in the accumulation of acetylcholine at cholinergic receptor sites, producing continuous stimulation of cholinergic fibers throughout the nervous systems.     

Rates of spontaneous reactivation and aging of acetylcholinesterase in human erythrocytes after inhibition by organophosphorus pesticides

The in vitro rates of spontaneous reactivation and aging in human erythrocyte acetylcholinesterase were studied after inhibition by a dimethoxy (R1R2) and diethoxy substituted (R1R2) organophosphate pesticide (OP) of general structure R1R2P(O)X. These have been compared with data for human plasma cholinesterase previously reported using a similar methodology. A significantly slower rate of aging for erythrocyte acetylcholinesterase was found compared to plasma cholinesterase, whether inhibited by dimethoxy or diethoxy substituted OPs. For diethoxy OPs the rate of spontaneous reactivation of the inhibited plasma enzyme was significantly slower than for the inhibited red cell enzyme. This acetylcholinesterase, and previously published plasma cholinesterase, data suggest that in practise a blood sample taken 30-40 h after significant acute OP exposure will still show inhibition in either plasma or erythrocyte cholinesterase when analysed, but that any inhibited plasma enzyme is more likely to be in the aged form. In contrast a substantial proportion of the erythrocyte acetylcholinesterase is found unaged and therefore sensitive to reactivation by oximes. Samples from an occupational exposure where depressions in plasma or erythrocyte cholinesterase activity from baseline measurements were reactivated ex vivo using the oxime 2-PAM support this hypothesis. These data also confirm that the plasma enzyme is a more sensitive than erythrocyte acetylcholinesterase as an indicator of OP exposure and thus the potential value of ex vivo oxime reactivation of erythrocyte acetylcholinesterase in a blood sample to indicate subclinical OP exposure may be limited. However, this study is too small to draw conclusions on the sensitivity of ex vivo oxime reactivation of acetylcholinesterase as a novel biomarker of excessive OP absorption. Given that there is a better relationship between anticholinergic symptoms and red cell acetylcholinesterase inhibition, and that the slower resynthesis rate of any aged or inhibited red cell enzyme may be interpretatively useful when venepuncture is delayed, it is suggested that red cell acetylcholinesterase activity does have a place in monitoring potential OP exposure.     

Levels of organophosphorus pesticides in medicinal plants commonly consumed in Iran

The frequent occurrence of pesticide residues in herbal materials was indicated by previous studies. In this study, the concentration of some of the organophosphorus pesticides including parathion, malathion, diazinon and pirimiphos methyl in different kinds of medicinal plants were determined. The samples were collected randomly from ten local markets of different areas of Iran. At the detection limit of 0.5 ng g^-1, parathion and pirimiphos methyl were not detected in any of the samples_. Some amounts of malathion and diazinon were found in Zataria, Matricaria chamomile, Spearmint and Cumin Seed samples while, the concentrations of target organophosphorus pesticides in Borage samples were below the detection limits of the methods which could be a result of intensive transformation of organophosphorus pesticides by Borage. In addition the organophosphorus pesticides were detected in all of the samples below the maximum residue levels (MRLs) proposed by the international organizations.     

A systematic review on organochlorine and organophosphorus pesticides content in water resources

Water resources pollution by uncontrolled usage of pesticides is one of the most critical health and environmental problems. However, to protect the ecosystems and to design strategy plans, it is essential to obtain information on type and amount of pesticide residues in water resources. The present study presents an overview of two important pesticides: organo-chlorine and organo-phosphorus in world-water resources. Based on literature review, most of studies have been conducted in Asian countries, and the amount that have been reported in water resources in these countries was more than the permissible level. The high-level of δ-HCH (Lindane) pesticides in groundwater with concentration ～82.21?μg/L was detected in China. In addition, the maximum measured concentration of Diazinon was detected in surface water in Iran with the value of ～768.91?μg/L. The overview of the studies shows widespread presence of pesticides in water resources, and it was found that surface waters were more contaminated by pesticides.     

A role for solvents in the toxicity of agricultural organophosphorus pesticides

Organophosphorus (OP) insecticide self-poisoning is responsible for about one-quarter of global suicides. Treatment focuses on the fact that OP compounds inhibit acetylcholinesterase (AChE); however, AChE-reactivating drugs do not benefit poisoned humans. We therefore studied the role of solvent coformulants in OP toxicity in a novel minipig model of agricultural OP poisoning. Gottingen minipigs were orally poisoned with clinically relevant doses of agricultural emulsifiable concentrate (EC) dimethoate, dimethoate active ingredient (AI) alone, or solvents. Cardiorespiratory physiology and neuromuscular (NMJ) function, blood AChE activity, and arterial lactate concentration were monitored for 12h to assess poisoning severity. Poisoning with agricultural dimethoate EC40, but not saline, caused respiratory arrest within 30 min, severe distributive shock and NMJ dysfunction, that was similar to human poisoning. Mean arterial lactate rose to 15.6 [SD 2.8] mM in poisoned pigs compared to 1.4 [0.4] in controls. Moderate toxicity resulted from poisoning with dimethoate AI alone, or the major solvent cyclohexanone. Combining dimethoate with cyclohexanone reproduced severe poisoning characteristic of agricultural dimethoate EC poisoning. A formulation without cyclohexanone showed less mammalian toxicity. These results indicate that solvents play a crucial role in dimethoate toxicity. Regulatory assessment of pesticide toxicity should include solvents as well as the AIs which currently dominate the assessment. Reformulation of OP insecticides to ensure that the agricultural product has lower mammalian toxicity could result in fewer deaths after suicidal ingestion and rapidly reduce global suicide rates.     

血药浓度检测在急性有机磷中毒患者救治中的应用

目的：探讨血药浓度检测在急性有机磷中毒（AOPP）患者救治中的应用。方法：选择医院急诊室AOPP患者采用气相色谱法进行血药浓度监测,研究血液毒物存在时间及血药浓度高低、血液毒物持续存在时间与血液胆碱酯酶（ChE）、氯磷定应用、住院时间的关系。结果：AOPP患者血液毒物持续存在时间多在3～7d,较传统观点明显延长;初始血药浓度较高和血液毒物持续存在时间较长者ChE恢复慢,氯磷定应用时间长、剂量大,患者病情重、住院时间长。结论：血药浓度监测对AOPP的病情判断、指导治疗、改善预后有重要意义。     

OpdA, a bacterial organophosphorus hydrolase, prevents lethality in rats after poisoning with highly toxic organophosphorus pesticides

Organophosphorus (OP) pesticides poison more than 3,000,000 people every year in the developing world, mostly through intentional self-poisoning. Advances in medical therapy for OP poisoning have lagged, and current treatment is not highly effective with mortality of up to 40% in even the most advanced Western medical facilities. Administration of a broadly active bacterial OP hydrolase to patients in order to hydrolyze OPs in circulation might allow current therapies to be more effective. The objective of this work was to evaluate the efficacy of a new recombinant bacterial OP hydrolase (OpdA), cloned from Agrobacterium radiobacter, in rat models of two chemically distinct but highly toxic and rapidly acting OP pesticides: dichlorvos and parathion. Without OpdA treatment, median time to death in rats poisoned with 3x LD(50) of dichlorvos or parathion was 6 min and 25.5 min, respectively. Administration of a single dose of OpdA immediately after dichlorvos resulted in 100% survival at 24h, with no additional antidotal therapy. After parathion poisoning, OpdA alone caused only a delay to death. However, an additional two doses of OpdA resulted in 62.5% survival at 24 h after parathion poisoning. In combination with pralidoxime therapy, a single dose of OpdA increased survival to 75% after parathion poisoning. Our results demonstrate that OpdA is able to improve survival after poisoning by two chemically distinct and highly toxic OP pesticides.     

19种有机磷农药对酵母D61.M菌株的诱变性

本文利用检测染色体丢失的酵母D61.M菌株对19种有机磷农药进行诱变性研究，并与本室过去利用其他两种诱变试验结果进行对比分析. 结果表明D61.M的检出率为89.5%(17/19)，即提示大多数有机磷农药可诱发非整倍体. 17种呈阳性的有机磷农药中有14种也能在体外微核试验中检出，它们可能单独引起非整倍体或兼能引起染色体断裂. 仅两种有机磷农药(乙硫磷，乙拌磷)单独在微核试验中检出而被D61.M漏检，提示只有少数有机磷农药单独具有断裂作用. 经进一步比较体内，体外微核试验及对甲基，乙基和其他有机磷农药的测试结果，提示D61.M对脱甲基以及“脱芳基”的能力最差.