Interaction of dialkyl(alpha-carbomethoxy-beta,beta,beta-trifluoro- ethyl) phosphates with mammalian esterases

The interaction of dialkyl (alpha-carbometoxy-beta,beta,beta-trifluoroethyl) phosphates (RO)2P(O) . OCH(CF3)COOMe (R = Me, Et, Pr, Pri, Bu, Bui, Am, Hex) (I-VIII) with human erythrocyte acetylcholinesterase, horse serum butyrylcholinesterase, pig liver carboxylesterase was studied and acute toxicity in mice was estimated. Compounds (I)-(VIII) were not hydrolyzed by carboxylesterase, slowly and irreversibly inhibited acetylcholinesterase (kII = 10(2)-10(4) M-1 X min-1) and more efficiently inhibited butyrylcholinesterase and carboxylesterase (kII = 10(3)-10(7) M-1 X min-1). The structure--antienzymatic activity relationships were investigated. With increasing of hydrophobicity of alkoxy groups, antienzymatic activity to butyrylcholinesterase and carboxylesterase ("sites of loss") rises equally and more significantly, than antiacetylcholinesterase activity (delta lg kII 1.0 and 2.4 for R = CH3 and C5H11 resp.). Branching at the alpha-position of alkoxy groups leads to sharp reducing of acetylcholinesterase and butyrylcholinesterase inhibition constants, the carboxylesterase inhibition mechanism becoming reversible. Multiple regression analysis (the Kubinyi model) showed that influence of steric hindrances is revealed at the phosphorylation stage. It was found that phosphates (I)-(VIII) possess low acute toxicity in mice (900-2000 mg/kg). The toxicity of this homologous series appears to be independent of the hydrophobicity. Role of esterases in toxicological effect of compounds (I)-(VIII) is discussed.     

Organophosphorus pesticide‐induced butyrylcholinesterase inhibition and potentiation of succinylcholine toxicity in mice

Succinylcholine is the most important rapid‐acting depolarizing muscle relaxant during anesthesia. Its desirable short duration of action is controlled by butyrylcholinesterase, the detoxifying enzyme. There are two reported cases of prolonged paralysis from succinylcholine in patients poisoned with the organophosphorus insecticides parathion and chlorpyrifos. The present study examines the possibility that other organophosphorus and methylcarbamate pesticides might also prolong succinylcholine action by inhibiting butyrylcholinesterase using mice treated intraperitoneally as a model and relating inhibition of blood serum hydrolysis of butyrylthiocholine to potentiated toxicity (mouse mortality). The organophosphorus plant defoliant tribufos (4 h pretreatment, 160 mg/kg) and organophosphorus plant growth regulator ethephon (1 h pretreatment, 200 mg/kg) potentiate the toxicity of succinylcholine by seven‐ and fourfold, respectively. Some other pesticides or analogs are more potent sensitizers for succinylcholine toxicity with threshold levels of 0.5, 1.0, 1.7, 8, 10, and 67 mg/kg for phenyl saligenin cyclic phosphonate, profenofos, methamidophos, tribufos, chlorpyrifos, and ethephon, respectively. Enhanced mortality from succinylcholine is generally observed when serum butyrylcholinesterase is inhibited 55–94%. Mivacurium, a related nondepolarizing muscle relaxant also detoxified by butyrylcholinesterase, is likewise potentiated by at least threefold on 4 hour pretreatment with tribufos (25 mg/kg) or profenofos (10 mg/kg). © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 113–118, 1999     

Comparative response of nestling European starlings and red-winged blackbirds to an oral administration of either dimethoate or chlorpyrifos.

Red-winged blackbird (Agelaius phoeniceus; blackbird) and European starling (Sturnus vulgaris; starling) nestlings were dosed with either 2.0 mg/kg body mass chlorpyrifos, 50.0 mg/kg body mass dimethoate, or a propylene glycol carrier in situ. Four growth measurements (body mass, culmen, tarsus, wing) were recorded from nestlings to determine if these organophosphorus compounds caused perturbations in development at sublethal concentrations. Blackbird nestlings were more sensitive to chlorpyrifos than starling nestlings were more sensitive to dimethoate than blackbird nestlings. This was in contrast to reported adult LD50 values where the reverse was true. Blackbird nestlings were more tolerant of a substantially higher concentration of dimethoate than the adult LD50. The sensitivity of starling nestlings to dimethoate was similar to adults. In contrast, juveniles of both species were more sensitive to chlorpyrifos than adults. After the initial 24 hr, surviving nestlings dosed with either chemical recovered and continued their development. Exposure to dimethoate caused significant depression in starling body mass during the initial 24 hr period. Survivors obtain body mass equal to controls within 48 hr post dosing. The research presented here demonstrates that the simple supposition that passerine nestlings are typically more sensitive to toxins than adults does not always hold true. It also indicates that sensitivity relationships among adults do not necessarily apply to their nestlings.     

Esterases as pesticide biomarkers in crayfish (Procambarus clarkii, Crustacea): tissue distribution, sensitivity to model compounds and recovery from inactivation

The specific activities of acetyl- and butyrylcholinesterase and carboxylesterase were assayed in the digestive gland and in nervous and muscle tissues of the crayfish Procambarus clarkii. Since acetylcholinesterase prevails in nervous tissue and carboxylesterase in digestive gland, they are proposed as biomarkers. Muscle had negligible activities of all esterases, and all tissues had a low butyrylcholinesterase activity. Esterases were mostly cytosolic in digestive gland and muscle, but membrane-bound in nervous tissue; use of Triton X-100 is not recommended due to its widely diverging effects in esterase assays. Phenylmethylsulphonylfluoride inhibited acetyl- and butyrylcholinesterase in extracts from all tissues, and in digestive gland only carboxylesterase. In digestive gland, tetra[monoisopropyl]-pyrophosphorotetramide inhibited all esterases with different sensitivities, while in muscle and nervous tissue it only partially inhibited all esterases. Carbamates inhibited 100-fold more strongly than organophosphates acetyl- and butyrylcholinesterase activities. Carboxylesterase was inhibited by carbaryl and chlorpyrifos, but not by eserine and malathion. In vitro conditions to evaluate recovery from inactivation of esterases by model pesticides were established for acetylcholinesterase and carboxylesterase. The new reactivation protocol could be useful as a biomarker of pesticide exposure to differentiate between dilution-reversible inhibitions, indicating carbamate exposure, from dilution-irreversible effect, attributed to organophosphate exposure.     

Pharmacological effects of a novel isosorbide-based butyrylcholinesterase inhibitor

Isosorbide-2-benzylcarbamate-5-benzoate, a novel butyrylcholinesterase inhibitor, shows interspecies variation in its inhibitory activity (IC(50) of 4.3 nM for human plasma butyrylcholinesterase, but 1.09 microM for mouse plasma butyrylcholinesterase). Stability studies revealed that this drug is resistant to hydrolysis by human plasma (no degradation in 1 h). However, it was found to undergo rapid degradation when incubated with mouse plasma or mouse liver homogenate, yielding benzyl carbamate and benzoic acid. The addition of the carboxylesterase inhibitor bis-(4-nitrophenyl) phosphate (BNPP) inhibited the degradation of the novel drug, indicating that it may be a substrate for both butyrylcholinesterase and carboxylesterase. The absence of carboxylesterase from human plasma explains the drug's stability in this medium. In vivo, pharmacodynamic studies on single doses of 1 mg/kg to na?ve male C57BL/6 mice revealed maximal plasma butyrylcholinesterase inhibition 20 min after intraperitoneal administration (approximately 60% inhibition) and 1 h after administration by gavage (approximately 45% inhibition). While this plasma butyrylcholinesterase inhibition was short-lived, the drug also penetrated the blood-brain barrier resulting in a slight (10-15%) but persistent (> or =72 h) reduction in brain butyrylcholinesterase activity.     

黄曲条跳甲对毒死蜱敏感性差异的生化机制

采用生物测定方法测定黄曲条跳甲Phyllotreta striolata(F.)2个室内试验种群(蔊菜试验种群、上海青试验种群)和1个田间自然种群对毒死蜱的LC50值。结果表明,蔊菜试验种群对毒死蜱的LC50值最低,为30.3459mg.L-1;田间自然种群对毒死蜱的LC50值最高,为77.8448mg.L-1,与蔊菜试验种群相比的敏感性指数为0.39。对不同种群黄曲条跳甲乙酰胆碱酯酶(AChE)、羧酸酯酶(CarE)、谷胱甘肽-S-转移酶(GSTs)的活性测定结果表明,黄曲条跳甲田间自然种群AChE活性最低,与菜试验种群、上海青试验种群相比,差异极显著(P>0.01);田间自然种群GSTs活性最高,与蔊菜试验种群、上海青试验种群相比,差异极显著(P>0.01);黄曲条跳甲蔊菜试验种群CarE活性最低,田间自然种群CarE活性最高,二者差异极显著(P>0.01);说明黄曲条跳甲对毒死蜱的敏感性下降可能与AChE活性的降低,与CarE、GSTs的活性提高有一定的关系。     

Tissue Carboxylesterases and Chlorpyrifos Toxicity in the Developing Rat

Young animals are more sensitive than adults to the neurotoxic effects of some organophosphorus insecticides. Many investigators attribute this difference in sensitivity to the immaturity of the detoxification capacity of preweanling rats. Chlorpyrifos [O,O-diethylO-(3,5,6-trichloro-2-pyridyl)phosphorothionate] is an organophosphorus insecticide that demonstrates considerable age-related sensitivity. The carboxylesterases are a group of related enzymes that detoxify organophosphorus insecticides by stoichiometrically binding these molecules before they can inhibit acetylcholinesterase. This study presents in vitro and in vivo evidence demonstrating that the carboxylesterases are critical for explaining the age-related sensitivity of chlorpyrifos. The data show that the fetal rat and the postnatal day 17 (PND17) rat pup have fewer molecules of carboxylesterase (less activity), less sensitive molecules of carboxylesterase, and a larger proportion of chlorpyrifos-insensitive molecules of carboxylesterase. An in vitro mixing experiment, using adult striatum as a source of acetylcholinesterase and liver homogenates as a source of carboxylesterase, demonstrates that the adult liver carboxylesterases are superior to the PND17 liver carboxylesterases for detoxifying chlorpyrifos. In the in vivo experiments the time course profiles of carboxylesterase and cholinesterase activity following a maximum tolerated dose of chlorpyrifos also suggest that the carboxylesterases of the PND17 rat were less capable of detoxifying chlorpyrifos. Carboxylesterase activity in the preweanling rat was not as severely inhibited as in the adult, but decrements in cholinesterase activity as a result of chlorpyrifos treatment were comparable. These in vitro and in vivo findings support the previously proffered postulate that the carboxylesterases are critical for determining the age-related sensitivity of chlorpyrifos. In addition, these detailed experiments allow us to propose that the detoxification potential of these enzymes is multifaceted, and depends on the (1) amount of activity (i.e., number of molecules), (2) affinity for the insecticide or metabolite, and (3) amount of carboxylesterase activity that is refractory to inhibition by the insecticide or metabolite.     

A novel, sensitive, reusable and low potential acetylcholinesterase biosensor for chlorpyrifos based on 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotubes gel

A novel, low potential and highly sensitive acetylcholinesterase (AChE) biosensor was developed based on 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotube composite gel thiocholine sensor. Composite gel promoted electron transfer reaction at a lower potential (+50 mV) and catalyzed electrochemical oxidation of thiocholine with high sensitivity. AChE was immobilized in sol-gel matrix that provides a good support for enzyme without any inhibition effect from the ionic liquid. The amount of immobilized enzyme and incubation time with chlorpyrifos were optimized. Chlorpyrifos could be determined in the range of 10(-8)-10(-6)M with a detection limit of 4 nM. Fast and efficient enzyme reactivation was obtained at low obidoxime concentration (0.1mM). Moreover, the biosensor exhibited a good stability and reproducibility and could be use for multiple determinations of pesticide with no loss of the enzyme activity.     

Protective effects of zinc in chlorpyrifos induced hepatotoxicity

The toxicological literature is replete with studies attempting to explain the mechanism of action of organophosphorus (OP) insecticides to their anticholinesterase activities, but not much is known about the metabolism and detoxification of these compounds. The goal of this study was to ascertain the toxic effects of chlorpyrifos, one of the most widely used OPs, on the liver of male rats and also to evaluate the protective potential of zinc in mediating its toxic effects. It was observed that chlorpyrifos (13.5 mg/kg body weight) treatment resulted in significant inhibition (p < 0.001) of serum and hepatic acetylcholinesterase (AChE) activities after 8 wk. However, zinc-treated (227 mg/L drinking water) animals resulted in significant normalization of the inhibited AChE activities. Similarly, a significant increase in the levels of various serum and liver marker enzymes (viz. alkaline phosphatase, aspartate aminotransferase [AST], and alanine aminotransferase [ALT]) was observed following treatment with chlorpyrifos. However, coadministration of zinc to these animals restored these enzymes to within normal limits, even though some increase in the activity of serum ALT and hepatic alkaline phosphatase still persisted at the end of the study. Chlorpyrifos treatment diminished serum and hepatic zinc levels significantly (p < 0.01 to p < 0.001) compared to normal control animals. Serum iron concentrations also plummeted significantly following chlorpyrifos treatment. On the contrary, serum copper levels were significantly increased (p < 0.01) in chlorpyrifos-treated animals, but they were normalized following zinc supplementation to the rats in this group. Interestingly, chlorpyrifos treatment resulted in elevated hepatic levels of copper, iron, and selenium, but zinc treatment could only partially restore the raised elemental concentrations. These data clearly demonstrate the potential role of zinc in mediating the toxic effects of chlorpyrifos, presumably because of their antioxidant properties and also their possible interaction with other trace elements in maintaining the cellular harmony.     

B型烟粉虱田间种群对毒死蜱和敌敌畏抗性的生化机制

通过增效剂生物测定和生化分析,探讨了采自福建省的B型烟粉虱Bemisia tabaci 6个田间种群对毒死蜱和敌敌畏抗性的生化机制。结果表明：与敏感品系SUD-S相比,6个田间种群对毒死蜱和敌敌畏分别具有54.53～78.43倍和6.23～11.25倍的抗性。TPP、PBO和DEM对毒死蜱的增效比分别为3.61～24.94倍、1.14～1.76倍和1.04倍,对敌敌畏的增效比分别为1.67～2.64倍、1.33～1.65倍和1.09倍,表明羧酸酯酶的解毒代谢在烟粉虱对毒死蜱的抗性中起着重要作用。烟粉虱抗性种群乙酰胆碱酯酶的K_m值是敏感品系的1.83～4.0倍,V _max值是敏感品系的0.34～0.62倍; 敏感品系乙酰胆碱酯酶的活性在底物浓度大于1.0 mmol/L时受抑制,抗性种群乙酰胆碱酯酶的活性在底物浓度大于16 mmol/L时受抑制;抗性种群乙酰胆碱酯酶对敌敌畏和毒死蜱的敏感度分别比敏感品系低119.92～161.33倍和10.11～14.24倍,表明烟粉虱田间抗性种群乙酰胆碱酯酶可能已发生了变构,由变构引起的乙酰胆碱酯酶不敏感是烟粉虱田间种群对毒死蜱和敌敌畏产生抗性的重要原因。结果提示,乙酰胆碱酯酶不敏感性和羧酸酯酶的解毒代谢在烟粉虱对毒死蜱的抗性中均起着重要作用,而乙酰胆碱酯酶不敏感性在对敌敌畏的抗性中起重要的作用,多功能氧化酶和谷胱甘肽S转移酶在烟粉虱对毒死蜱和敌敌畏抗性中所起的作用不大。     

Immobilization of rat brain acetylcholinesterase on ZnS and poly(indole-5-carboxylic acid) modified Au electrode for detection of organophosphorus insecticides

A novel, highly sensitive amperometric biosensor for detection of organophosphorus (OP) compounds has been constructed, based on rat brain acetylcholinesterase (AChE) immobilized onto nanocomposite of ZnS-nanoparticles (ZnSNPs) and poly(indole-5-carboxylic acid) electrodeposited on Au electrode. In the presence of acetylthiocholine chloride (ATCl) as a substrate, ZnSNPs promoted electron transfer reactions at a lower potential and catalyzed electrochemical oxidation of enzymatically formed thiocholine, thus increasing detection sensitivity. Under optimum conditions (phosphate buffer, pH 7.5 and 30°C), the inhibition of AChE by malathion and chlorpyrifos was proportional to their concentrations in the range, 0.1-50nM and 1.5-40nM, respectively. The biosensor determined malathion and chlorpyrifos in spiked tap water samples with a acceptable accuracy (95-100%). The enzyme electrode had long-storage stability (50% retention of initial activity within 2 months, when stored at 4°C).     

Quercetin plays protective role in oxidative induced apoptotic events during chronic chlorpyrifos exposure to rats

Chlorpyrifos (CPF), an organophosphate insecticide has a wider application throughout the world to protect agricultural crops and vegetables from insects. Polyphenolic compounds are considered as beneficial against toxicities induced by organophosphates. The present study was conducted to understand the neuroprotective role of quercetin in chlorpyrifos‐induced apoptotic events in rats. Twenty‐four male Sprague Dawley rats weighing 170 to 200 g were divided into four groups viz: Control, chlorpyrifos treated (13.5 mg/kg body wt. alternate day), quercetin treated (50 mg/kg body wt. every day) and combined chlorpyrifos + quercetin treated. All the treatments were carried out for a total duration of 60 days. Protein carbonyl content and acetylcholinesterase activity were estimated in serum along with cerebrum and cerebellum to ascertain neurotoxicity. Further, for appraisal of neurodegeneration as a consequence of apoptosis, protein expressions of Bcl‐2, Bax, cytochrome c, caspase‐8, and caspase‐9 were assessed. The results showed that protein carbonyl contents were markedly increased in both serum and brain tissues (cerebrum and cerebellum) of chlorpyrifos‐treated rats when compared with control group and were appreciably improved upon simultaneous supplementation with quercetin. Further, chlorpyrifos treatment revealed a significant decrease in the enzyme activity of acetylcholinesterase in serum as well as in cerebrum and cerebellum, which however was increased upon concomitant treatment with quercetin. In chlorpyrifos‐treated animals, we have observed a significant decrease in the protein expression level of Bcl‐2, but a remarkable increase in the expression levels of Bax, cytochrome c, caspase‐8, and caspase‐9 in both cerebrum and cerebellum. Interestingly, when chlorpyrifos‐treated animals were supplemented with quercetin, a significant increase in the expression of Bcl‐2 and an appreciable decline in the expression levels of Bax, cytochrome c, caspase‐8, and caspase‐9 was observed. In conclusion, the present study advocates that quercetin may prove to be a useful candidate in containing the oxidative‐induced apoptotic events during chlorpyrifos exposure.     

Differential modulation of organophosphate-sensitive muscarinic receptors in rat brain by parathion and chlorpyrifos

We previously reported similar levels of brain cholinesterase inhibition but marked differences in toxicity following acute maximum tolerated doses of the organophosphate pesticides parathion and chlorpyrifos. Because extensive acetylcholinesterase inhibition often induces compensatory changes in cholinergic receptor populations, we compared the effects of parathion and chlorpyrifos on brain muscarinic receptors. Adult male rats were treated with vehicle or the maximum tolerated dose of parathion (18 mg/kg, sc) or chlorpyrifos (279 mg/kg, sc) and observed for signs of acute toxicity. Similarly treated animals were sacrificed at 2, 7, or 14 days after treatment for measurement of cholinesterase activity and binding to the nonselective muscarinic antagonist [³H]quinuclidinyl benzilate, the M2-preferential antagonist [³H]AFDX-384, and the high-affinity agonist [³H]cis-methyldioxolane. More acute toxicity was noted after parathion treatment. Both insecticides caused similar levels (> 85%) of maximal cholinesterase inhibition and reductions (up to 55%) in atropine-sensitive quinuclidinyl benzilate binding (i.e., total muscarinic receptors) and [³H]AFDX-384 binding in cortex and striatum. Parathion also reduced, whereas chlorpyrifos increased, total muscarinic receptor binding and [³H]AFDX-384 binding in the cerebellum. When tissues were preincubated with paraoxon (10 μM), radiolabeling of a subset of quinuclidinyl benzilate binding sites was blocked and the apparent densities of these organophosphate-sensitive receptors in all three tissues were decreased (16% maximal) by parathion but increased (up to 37%) by chlorpyrifos. Similarly, parathion decreased whereas chlorpyrifos increased [³H]cis-methyldioxolane binding sites in all three brain regions. We propose that differential modulation of these organophosphate-sensitive muscarinic receptors contributes to differences in acute toxicity following exposure to these pesticides.     

Dimethoate,fenvalerate and their mixture affects Hylyphantes graminicola (Araneae: Linyphiidae) adults and their unexposed offspring

• 1 Mixtures of organophosphorus and pyrethroid insecticides are widely used to combat resistance in agricultural pests, although few studies have been conducted on the effects of pesticide mixtures on beneficial nontarget organisms.
• 2 In the present study, we exposed adult females (F₀) of Hylyphantes graminicola (Araneae: Linyphiidae) to fenvalerate, dimethoate and their commercially available 1 : 1 mixture (by mass). We investigated the acute toxicity of these pesticides to the exposed adults, as well as sublethal effects on reproduction and acetylcholinesterase and carboxylesterase activity. We also studied the effects of parental exposure on the size, development and enzyme activity of unexposed offspring.
• 3 All three formulations were acutely toxic to H. graminicola, with synergism between dimethoate and fenvalerate leading to greater toxicity in the 1 : 1 mixture than for the two insecticides alone. The sublethal effects of direct pesticide exposure were a reduction in acetylcholinesterase and carboxylesterase activity and a reduction in the number of egg sacs produced by exposed spiders relative to the control spiders. The unexposed offspring of the fenvalerate and mixture exposed spiders were smaller and took longer to mature than the control spiders. Offspring of all exposed spiders also had significantly reduced carboxylesterase activity relative to control spiders.
• 4 We concluded that the effects of parental exposure on the offspring were likely to increase their susceptibility to future pesticide exposures, and reduce the capacity of this spider to serve as a pest control agent.

     

Acetylcholinesterase mediated susceptibility of soldiers and workers of formosan subterranean termite (Isoptera: Rhinotermitidae) to chlorpyrifos

Target site studies were undertaken to examine the difference in susceptibility of Formosan subterranean termite, Coptotermes formosanus Shiraki, workers and soldiers to chlorpyrifos. Workers exhibited significantly greater acetylcholinesterase activity per insect than soldiers (118.63 +/- 48.51 versus 47.98 +/- 22.59 mOD/min/insect equivalent). Likewise, enzyme activity (mean +/- SD) per milligram of protein was greater in workers than soldiers (440.30 +/- 267.43 versus 311.53 +/- 149.83 mOD/min/mg protein). The enzyme of soldiers was more sensitive to the acetylcholinesterase (AChE) inhibitors eserine and chlorpyrifos-oxon than that of workers. The I50s of chlorpyrifos-oxon were 2.66 and 4.59 nM for soldiers and workers, respectively, whereas the I50s of eserine were 16.56 and 25.41 nM for soldiers and workers, respectively. The amount of protein was significantly higher in workers than in soldiers with mean values of 0.270 +/- 0.102 and 0.154 +/- 0.054 mg/insect equivalent, respectively. We suggest that the differential response of workers and soldiers to chlorpyrifos may be due to the difference in AChE sensitivity to inhibition and the amount of protein between them.     

Biochemical and proteomic effects in Procambarus clarkii after chlorpyrifos or carbaryl exposure under sublethal conditions

In vivo effects of two sublethal doses of chlorpyrifos and carbaryl were studied in Procambarus clarkii after 2 and 7 days of exposure, and after pesticide removal. Chlorpyrifos inhibited carboxylesterase activity in a concentration-dependent manner, but acetylcholinesterase was less sensitive. Compared with chlorpyrifos, carbaryl had a less marked effect on esterase activity. The effects of selected pesticides on biotransformation or oxidative stress biomarkers were contradictory. Chlorpyrifos lowered ethoxyresorufin-O-deethylase (EROD), catalase and oxidized glutathione (GSSG) levels but raised glutathione-S-transferase activity, while carbaryl raised EROD, catalase and glutathione-S-transferase, but lowered glutathione peroxidase and reduced glutathione (GSH) levels. The effects on protein expression patterns depending on pesticide type and the tissue used for analysis were studied in parallel by 2-DE. In gill and nervous tissue about 2000 spots (pI 4–7) were resolved, with quite different expression patterns. Chlorpyrifos altered 72 proteins, mostly in nervous tissue, and carbaryl 35, distributed evenly between organs. Several specific spots were selected as specific protein expression signatures for chlorpyrifos or carbaryl exposure in gills and nervous tissue, respectively.     

Effect of in vivo nicotine exposure on chlorpyrifos pharmacokinetics and pharmacodynamics in rats

Routine use of tobacco products may modify physiological and metabolic functions, including drug metabolizing enzymes, which may impact the pharmacokinetics of environmental contaminants. Chlorpyrifos is an organophosphorus (OP) insecticide that is bioactivated to chlorpyrifos-oxon, and manifests its neurotoxicity by inhibiting acetylcholinesterase (AChE). The objective of this study was to evaluate the impact of repeated nicotine exposure on the pharmacokinetics of chlorpyrifos (CPF) and its major metabolite, 3,5,6-trichloro-2-pyridinol (TCPy) in blood and urine and also to determine the impact on cholinesterase (ChE) activity in plasma and brain. Animals were exposed to 7-daily doses of either 1 mg nicotine/kg or saline, and to either a single oral dose of 35 mg CPF/kg or a repeated dose of 5 mg CPF/kg/day for 7 days. Groups of rats were then sacrificed at multiple time-points after receiving the last dose of CPF. Repeated nicotine and CPF exposures resulted in enhanced metabolism of CPF to TCPy, as evidenced by increases in the measured TCPy peak concentration and AUC in blood. However, there was no significant difference in the amount of TCPy (free or total) excreted in the urine within the first 24-h post last dose. The extent of brain acetylcholinesterase (AChE) inhibition was reduced due to nicotine co-exposure consistent with an increase in CYP450-mediated dearylation (detoxification) versus desulfuration. It was of interest to note that the impact of nicotine co-exposure was experimentally observed only after repeated CPF doses. A physiologically based pharmacokinetic model for CPF was used to simulate the effect of increasing the dearylation V_max based upon previously conducted in vitro metabolism studies. Predicted CPF-oxon concentrations in blood and brain were lower following the expected V_max increase in nicotine treated groups. These model results were consistent with the experimental data. The current study demonstrated that repeated nicotine exposure could alter CPF metabolism in vivo, resulting in altered brain AChE inhibition.     

Inhibition of forskolin-stimulated cAMP formation in vitro by paraoxon and chlorpyrifos oxon in cortical slices from neonatal, juvenile, and adult rats

Parathion (PS) and chlorpyrifos (CPF) are organophosphorus insecticides, which elicit toxicity following biotransformation to the potent acetylcholinesterase inhibitors, paraoxon (PO) and chlorpyrifos oxon (CPO). Both oxons have also been shown to interact directly with muscarinic receptors coupled to inhibition of adenylyl cyclase. Immature animals are more sensitive than adults to the acute toxicity of PS and CPF but little is known regarding possible age-related differences in interactions between these toxicants and muscarinic receptors. We compared the inhibition of forskolin-stimulated cAMP formation by PO and CPO (1 nM-1 mM) in vitro in brain slices from 7-, 21-, and 90-day-old rats to the effects of well-known muscarinic agonists, carbachol and oxotremorine (100 microM). Both agonists inhibited cAMP formation in tissues from all age groups and both were more effective in adult and juvenile (20-26% inhibition) than in neonatal (12-13% inhibition) tissues. Atropine (10 microM) completely blocked agonist-induced inhibition in all cases. PO maximally inhibited (37-46%) cAMP formation similarly in tissues from all age groups, but atropine blocked those effects only partially and only in tissues from 7-day-old rats. CPO similarly inhibited cAMP formation across age groups (27-38%), but ATR was partially effective in tissues from all three age groups. Both oxons were markedly more potent in tissues from younger animals. We conclude that PO and CPO can directly inhibit cAMP formation through muscarinic receptor-dependent and independent mechanisms and that the developing nervous system may be more sensitive to these noncholinesterase actions.     

Synthesis,in-vitro cholinesterase inhibition,in-vivo anticonvulsant activity and in-silico exploration of N-(4-methylpyridin-2-yl)thiophene-2-carboxamide analogs

In our current research, a diverse effect of acetylcholinesterase inhibitors was studied on BALB-C mice by using pentylenetetrazole (PTZ) seizure model. A series of carboxamide analogs (4a–4i) have been synthesized via Suzuki coupling reaction in moderate to good yields (35–84%). To study the efficacy of the synthesized compounds against AD, in-vitro inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was performed. A number of compounds showed inhibition in low micromolar range. Subsequently, these compounds were evaluated for anticonvulsive effects in BALB-C mice by using pentylenetetrazole (PTZ) seizure model. The compound 4e displayed potential anticonvulsive effect and displayed 50% and 80% protection from mortality at the dose of 10 mg/kg, and 30 mg/kg respectively. The compound 4h showed some protection (33%) from mortality at 10 mg/kg and was not further explored based on non-significant delay in onset of myoclonic seizures. While, other compounds from the series did not show any anticonvulsive activity. To rationalize the observed biological activity, we performed docking studies against AChE and BChE targets. To explore the rationale of the mechanism of in-vivo anticonvulsant activity, docking studies were performed on GABAergic receptors. Moreover, in order to establish a relationship between physiochemical data of the synthesized compounds and their in-vivo performance, we employed in-silico pharmacokinetic predictions. Our in-silico predictions suggest that the plasma protein binding, low to moderate blood brain barrier penetration and low solubility are the main attributes of low in-vivo performance.     

Anticholinesterase effect and toxicity of bis(trichloromethyl) sulfone (N-1386 Biocide) in rats

The oral LD50 for bis(trichloromethyl) sulfone (N-1386 Biocide) in male rats was 691 mg/kg. Deaths occurred 1-5 days after treatment and signs of toxicity suggestive of an anticholinesterase effect were noted. However, neither plasma cholinesterase nor brain acetylcholinesterase was inhibited 2, 4 or 24 hours after a single, oral dose of 500 mg/kg. Atropine (300 mg/kg, s.c.) or scopolamine (670 mg/kg, s.c.) pretreatments did not protect against the acute lethality of bis(trichloromethyl) sulfone although signs of toxicity were alleviated by both pretreatments. Bis(trichloromethyl) sulfone produced in vitro inhibition of rat plasma cholinesterase and brain acetylcholinesterase. The inhibition was competitive in brain. IC50's for these 2 enzymes were 8 microM in plasma and 25 microM in brain. In summary, bis(trichloromethyl) sulfone produced in vitro cholinesterase inhibition not demonstrated in vivo. Doses of anticholinergic compounds that ameliorated many toxic signs did not protect against lethality produced by bis(trichloromethyl) sulfone.