Oxon Analogues Research Articles

The influence of the model pesticides parathion and paraoxon on human cytochrome P450 and associated oxygenases in HepaRG cells

Introduction Intentional and unintentional organophosphorus pesticide exposure is a public health concern. Organothiophosphate compounds require metabolic bioactivation by the cytochrome P450 system to their corresponding oxon analogues to act as potent inhibitors of acetylcholinesterase. It is known that interactions between cytochrome P450 and pesticides include the inhibition of major xenobiotic metabolizing cytochrome P450 enzymes and changes on the genetic level. Methods In this in vitro study, the influence of the pesticides parathion and paraoxon on human cytochrome P450 and associated oxygenases was investigated with a metabolically competent cell line (HepaRG cells). First, the viability of the cells after exposure to parathion and paraoxon was evaluated. The inhibitory effect of both pesticides on cytochrome P450 3A4, which is a pivotal enzyme in the metabolism of xenobiotics, was examined by determining the dose-response curve. Changes on the transcription level of 92 oxygenase associated genes, including those for important cytochrome P450 enzymes, were evaluated. Results The exposure of HepaRG cells to parathion and paraoxon at concentrations up to 100 µM resulted in a viability of 100 per cent. After exposure for 24 hours, pronounced inhibition of cytochrome P450 3A4 enzyme activity was shown, indicating 50 per cent effective concentrations of 1.2 µM (parathion) and 2.1 µM (paraoxon). The results revealed that cytochrome P450 involved in parathion metabolism were significantly upregulated. Discussion Relevant changes of the cytochrome P450 3A4 enzyme activity and significant alteration of genes associated with cytochrome P450 suggest an interference of pesticide exposure with numerous metabolic processes. The major limitations of the work involve the use of a single pesticide and the in vitro model as surrogate to human hepatocytes. Conclusion The data of this study might be of relevance after survival of acute, life-threatening intoxications with organophosphorus compounds, particularly for the co-administration of drugs, which are metabolized by the affected cytochrome P450.

Acetylcholinesterase inhibition in electric eel and human donor blood: an in vitro approach to investigate interspecies differences and human variability in toxicodynamics

In chemical risk assessment, default uncertainty factors are used to account for interspecies and interindividual differences, and differences in toxicokinetics and toxicodynamics herein. However, these default factors come with little scientific support. Therefore, our aim was to develop an in vitro method, using acetylcholinesterase (AChE) inhibition as a proof of principle, to assess both interspecies and interindividual differences in toxicodynamics. Electric eel enzyme and human blood of 20 different donors (12 men/8 women) were exposed to eight different compounds (chlorpyrifos, chlorpyrifos-oxon, phosmet, phosmet-oxon, diazinon, diazinon-oxon, pirimicarb, rivastigmine) and inhibition of AChE was measured using the Ellman method. The organophosphate parent compounds, chlorpyrifos, phosmet and diazinon, did not show inhibition of AChE. All other compounds showed concentration-dependent inhibition of AChE, with IC50s in human blood ranging from 0.2–29 µM and IC20s ranging from 0.1–18 µM, indicating that AChE is inhibited at concentrations relevant to the in vivo human situation. The oxon analogues were more potent inhibitors of electric eel AChE compared to human AChE. The opposite was true for carbamates, pointing towards interspecies differences for AChE inhibition. Human interindividual variability was low and ranged from 5–25%, depending on the concentration. This study provides a reliable in vitro method for assessing human variability in AChE toxicodynamics. The data suggest that the default uncertainty factor of ~ 3.16 may overestimate human variability for this toxicity endpoint, implying that specific toxicodynamic-related adjustment factors can support quantitative in vitro to in vivo extrapolations that link kinetic and dynamic data to improve chemical risk assessment.

The Neuronal Cytoskeleton as a Potential Target in the Developmental Neurotoxicity of Organophosphorothionate Insecticides

Phosphorothionates are toxicologically the most important class of organophosphorus ester (OP) insecticides. Phosphorothionates are metabolically converted in vivo to their oxon analogues. These oxon metabolites can bind and inhibit acetylcholinesterase, thus causing acute cholinergic neurotoxicity. Oxon binding to the same target may also be partly responsible for manifestation of the 'intermediate syndrome'. More recent evidence suggests that the oxons may be also capable of inducing developmental neurotoxicity. The neuronal cytoskeleton may represent a potential target for the developmental neurotoxicity of the oxons because of its vital importance in many stages of normal neurodevelopment. Data obtained in the last five years and critically reviewed here indicate that the oxon metabolites, at concentrations that can be attained in vivo, exert potent effects on the neuronal cytoskeleton disrupting all three cytoskeletal networks. This disruption is expressed at the level of cytoskeletal protein expression, intracellular distribution, post-translational modification, cytoskeletal dynamics and function and may involve effects on both neuronal and glial cells. These effects are not secondary to other changes but may constitute primary effects of the oxons, as these compounds have been shown to be capable of covalently binding to and organophosphorylating multiple sites on tubulin and actin. Analogous studies must be extended to include other neurodevelopmentally important cytoskeletal proteins, such as neurofilament heavy chain, and tau, which are known to contain unusually high numbers of phosphorylatable sites and to establish whether organophosphorylation by the oxons takes place at sites where neurodevelopmentally relevant, endogenous, reversible phosphorylation is known to occur.

Degradation study of selected organophosphorus insecticides in natural waters

The degradation of 15 organophosphorus insecticides was studied in drinking, ground, and surface waters under different laboratory-controlled and environmental conditions. Surface waters originated from rivers Savinja (near the city of Celje) and Kamniska Bistrica (at the spring), which both belong to the Danube river basin. Groundwater was collected from wells (70 m deep) in Ljubljana, which are the direct source of drinking water for the capital. These matrices were selected on the basis of their different chemical composition and microbial activity. Major factors influencing the degradation were determined, such as temperature, oxygen, sunlight, pH, and type of water. The degradation of atrazine, present in many water sources in Slovenija, was followed simultaneously as a reference under the same conditions. The degradation kinetics was followed by gas chromatography with mass-selective detection, which also allowed the identification of some degradation by-products, such as oxon analogues paraoxon, dyfoxon, malaoxon, phenyl-methyl sulfoxide, fenthion sulfone, phorate sulfoxide, and phorate sulfone. The results show that the half-lives of the selected organophosphorus insecticides varied from 4 to 192 days or more, depending on the water source and experimental conditions. As a result, kinetically constants and half-lives were calculated for every tested insecticide, and major degradation products were determined.

Cutinase Inhibition by Means of Insecticidal Organophosphates and Carbamates. 3. Oxidation of Phosphorothionates by Chloroperoxidase from Caldariomyces fumago

Chloroperoxidase (CPO) from Caldariomyces fumago combined with hydrogen peroxide and chloride proved to be most efficient for the transformation of organophosphorothionate pesticides, i.e., chlorpyrifos, chlorpyrifos-methyl, parathion, and parathion-methyl, into their more potent serine esterase inhibiting oxon analogues. Following CPO pre-oxidation steps, the detection limit of a recently described spectrophotometric cutinase assay could be increased by about 2 orders of magnitude as a consequence of increased inhibition rates of the organophosphates. This type of enzymatic oxidation is easier to perform and more efficient, as compared to bromine or N-bromosuccinimide, used for acetylcholine esterase (AChE) assay in water analyses, but is insufficient for complex matrices such as plant sample extracts. The performance of a complete assay, including sample preparation, oxidation, and inhibition, takes about 3 h. Performing oxidations of organophosphorus compounds, two significant anomalies were observed. Upon CPO oxidation, chlorpyrifos-methyl showed a very strong cutinase inhibition as compared to the corresponding oxon standard, and oxidized malathion, contrarily to malaoxon, revealed cutinase inhibition, which however obeyed a reversible reaction mechanism in contrast to the usually irreversible reactions of organophosphates. Except for methomyl, no significant effects of CPO oxidation on the inhibition strength of insecticidal carbamates could be detected. The applicability of the assay was tested with fruit samples spiked with chlorpyrifos at 0.2-0.5 mg/kg, thereby regarding the role of the latter as the pesticide detected most often in fruits. Mean recoveries ranged between 30-50%. An enhanced recovery of 84% was obtained for an apple juice sample spiked with parathion-methyl (0.5 mg/L).

Photodegradation of organophosphorus insecticides – Investigations of products and their toxicity using gas chromatography–mass spectrometry and AChE-thermal lens spectrometric bioassay

Four organophosphorus compounds: azinphos-methyl, chlorpyrifos, malathion and malaoxon in aqueous solution were degraded by using a 125 W xenon parabolic lamp. Gas chromatography–mass spectrometry (GC–MS) was used to monitor the disappearance of starting compounds and formation of degradation products as a function of time. AChE-thermal lens spectrometric bioassay was employed to assess the toxicity of photoproducts. The photodegradation kinetics can be described by a first-order degradation curve C = C 0e − kt , resulting in the following half lives: 2.5 min for azinphos-methyl, 11.6 min for malathion, 13.3 min for chlorpyrifos and 45.5 min for malaoxon, under given experimental conditions. During the photoprocess several intermediates were identified by GC–MS suggesting the pathway of OP degradation. The oxidation of chlorpyrifos results in the formation of chlorpyrifos-oxon as the main identified photoproduct. In case of malathion and azinphos-methyl the corresponding oxon analogues were not detected. The formation of diethyl (dimethoxy-phosphoryl) succinate in traces was observed during photodegradation of malaoxon and malathion. Several other photoproducts including trimethyl phosphate esters, which are known to be AChE inhibitors and 1,2,3-benzotriazin-4(3H)-one as a member of triazine compounds were identified in photodegraded samples of malathion, malaoxon, and azinphos-methyl. Based on this, two main degradation pathways can be proposed, both result of the (P–S–C) bond cleavage taking place at the side of leaving group. The enhanced inhibition of AChE observed with the TLS bioassay during the initial 30 min of photodegradation in case of all four OPs, confirmed the formation of toxic intermediates. With the continuation of irradiation, the AChE inhibition decreased, indicating that the formed toxic compounds were further degraded to AChE non-inhibiting products. The presented results demonstrate the importance of toxicity monitoring during the degradation of OPs in processes of waste water remediation, before releasing it into the environment.

Abiotic transformation of chlorpyrifos to chlorpyrifos oxon in chlorinated water

In vivo transformation of chlorpyrifos to chlorpyrifos oxon is believed to be a prerequisite for this insecticide to display acute toxicity to organisms. We discovered that active chlorine dispersed in water causes the rapid abiotic transformation of chlorpyrifos to chlorpyrifos oxon. The proposed mechanism for the transformation is an electrophilic attack by hypochlorous acid (HOCl) on the thion (P=S) double bond of chlorpyrifos followed by desulfuration of chlorpyrifos to its oxon analog. The pH of a Cl-aqueous solution controls the speciation of HOCl and hypochlorite (OCl-) in water and thereby influences the transformation process. Chlorpyrifos oxon is a potent anticholinesterase that is about 1,000 times more toxic than chlorpyrifos. Because chlorination is commonly used for treatment of domestic water supplies, the findings raise a new concern about the safety of domestic use of chlorpyrifos products.

Aerobic Aquatic Metabolism of Fenitrothion and Its Oxon Analog in Water-Sediment Systems

フェニトロチオン及びそのオキソン体のフランスの湖水及び日本の池水-底質系での好気的な代謝試験を行った. 系への通気方法がフェニトロチオンの代謝経路に与える影響を見るために, 水層中に穏やかに通気するかまたは水表面上に空気を流す方法で実験を行った. いずれの方法においても代謝経路はほぼ同じであったが水-底質間の放射能分布に若干の違いが認められた. 水中通気による嫌気雰囲気の減少から, フェノール体と二酸化炭素の生成量が増加し還元的代謝が減少した. フランスと日本の水-底質系でのフェニトロチオンの代謝に顕著な差異は認められず主にP-O-アリール結合の開裂によるフェノール生成, ニトロ基のアミノ基への還元とそれに続くアセチル化により分解された. オキソン体の生成 (<1%) は主要な分解経路でばなく, 速やかにP-O-アリール結合が開裂しフェノール体に分解された.

Analysis of the Time-Dependent Acute Aquatic Toxicity of Organophosphorus Pesticides: The Critical Target Occupation Model

A model is presented for the acute toxicity of organophosphorus (OP) pesticides belonging to the class of phosphorothionates. The acute toxicity of these pesticides is governed by the irreversible inhibition of the enzyme acetylcholinesterase (AChE), after their metabolic activation to oxon analogues. The model is based on the idea that, for chemicals exhibiting an irreversible receptor interaction, mortality is associated with a critical amount of “covalently occupied” target sites, i.e., the “critical target occupation” (CTO). For a given compound and species, this CTO is associated with a critical time-integrated concentration of the oxon analogue in the target tissue, which can be modeled by the critical area under the curve (CAUC) that describes the time−concentration course of the phosphorothionate in the aqueous phase or in the entire aquatic organism. In contrast to the classical critical body residue (CBR) model, the CTO model successfully describes the 1−14-d LC50(t) data of several phosphorothi...

Characterization of the elevated esterase-associated insecticide resistance mechanism in Nilaparvata lugens (Stal) and other planthopper species

Insecticide-resistant strains of the brown planthopper, Nilaparvata lugens, from four locations all had a single diffuse elevated esterase band on native polyacrylamide gel electrophoresis. The white backed planthopper Sogatella furcifera had two elevated esterases with lower relative mobilities than the N. lugens esterase, while the insecticide-susceptible N. bakeri and the grass-associated sibling species of N. lugens sensu lato all had a single low intensity staining esterase with a lower relative mobility than the resistance associated N. lugens esterase. All the esterases were inhibited by pre-incubation with 0.1 mM paraoxon, but were not affected by permethrin up to its solubility limit, indicating their possible role in organophosphorus, but not pyrethroid insecticide, resistance. Partial purification of the elevated esterase from insecticide resistant Sri Lankan N. lugens showed that despite its diffuse nature on gels it purified as a single isoform, with a specific activity of 5.85 mumol min- 1 mg- 1 and an estimated molecular weight of approximately 60 kDa. Insecticide resistance was conferred by this elevated esterase through rapid binding and slow turnover of the carbamate or the insecticidally active oxon analogues of the phosphorothioates, i.e. sequestration rather than metabolism is the primary resistance mechanism. In contrast to earlier studies on the elevated esterases of N. lugens from Japan and the Philippines, we were unable to detect any malathion metabolism by this esterase, nor did malathion inhibit the esterase, although it was sensitive to inhibition by malaoxon. The elevated N. lugens esterase did not cross-react with antisera raised to the elevated mosquito or aphid esterases. The efficacy of the partially purified N. lugens esterase in insecticide sequestration and turnover is compared with other well characterized amplified esterases from mosquitoes and aphids.

Methyl parathion activation by a partially purified rat brain fraction

Organophosphorus pesticides are one of the most commonly used insecticide classes. They act through a potent inhibition of acetylcholinesterase (AChE). Many of them must undergo transformation into the corresponding oxon analogs to inhibit AChE. This study showed that a brain tissue subfraction transformed methyl parathion ( O, O-dimethyl O- p-nitrophenyl phosphorothioate) in vitro. Methyl parathion activation was assayed by solvent extraction of the products followed by HPLC and GC-MS analyses and, indirectly, by the inhibition of AChE present in the incubation mixture. The lack of impairment of AChE after 2 h of incubation of the brain subfraction with methyl parathion and, alternatively, with NADPH, CO, SKF 525-A, piperonyl butoxide or nitrogen indicated that this brain subfraction transformed methyl parathion without the involvement of a mixed-function oxidative pathway.The results from HPLC analysis did not show a peak corresponding to methyl paraoxon ( O, O-dimethyl O- p-nitrophenylphosphate), but showed the production of an unidentified peak which eluted nearby standard methyl parathion (retention times of 10.65 and 8.86 min, respectively). GC-MS analysis suggested that the unidentified product could be a methyl parathion isomer.

Recent research on problems in the use of urea as a nitrogen fertilizer

Recent research on the NH3 volatilization, NO 2 - accumulation, and phytotoxicity problems encountered in the use of urea fertilizer is reviewed. This research has shown that the adverse effects of urea fertilizers on seed germination and seedling growth in soil are due to NH3 produced through hydrolysis of urea by soil urease and can be eliminated by addition of a urease inhibitor to these fertilizers. It also has shown that the leaf burn commonly observed after foliar fertilization of soybean with urea results from accumulation of toxic amounts of urea in soybean leaves rather than formation of toxic amounts of NH3 through hydrolysis of urea by leaf urease. It further showed that this leaf burn is accordingly increased rather than decreased by addition of a urease inhibitor to the urea fertilizer applied. N-(n-butyl)thiophosphoric triamide (NBPT) is the most effective compound currently available for retarding hydrolysis of urea fertilizer in soil, decreasing NH3 volatilization and NO 2 - accumulation in soils treated with urea, and eliminating the adverse effects of urea fertilizer on seed germination and seedling growth in soil. NBPT is a poor inhibitor of plant or microbial urease, but it decomposes quite rapidly in soil with formation of its oxon analog N-(n-butyl) phosphoric triamide, which is a potent inhibitor of urease activity. It is not as effective as phenylphosphorodiamidate (PPD) for retarding urea hydrolysis and ammonia volatilization in soils under waterlogged conditions, presumably because these conditions retard formation of its oxon analog. PPD is a potent inhibitor of urease activity but it decomposes quite rapidly in soils with formation of phenol, which is a relatively weak inhibitor of urease activity. Recent studies of the effects of pesticides on transformations of urea N in soil indicate that fungicides have greater potential than herbicides or insecticides for retarding hydrolysis of urea and nitrification of urea N in soil.

Metabolism of the urease inhibitor n-( n -butyl)thiophosphoric triamide (nbpt) in soils

An experiment was conducted to relate the effectiveness of N-( n-butyl)thiophosphoric triamide (NBPT) and its oxon analog N-( n-butyl)phosphoric triamide (BNPO) in controlling urea hydrolysis in soils to their corresponding soil concentrations. Both compounds were applied to an acid soil (pH 4.9) and to the same soil that had been neutralized (pH 7.1) by long-term liming or by the recent application of Ca(OH) 2. Hydrolysis of urea applied with the inhibitors was monitored along with the disappearance of the compounds themselves. Both compounds controlled urea hydrolysis much more effectively in the neutral soils than in the acid soil. HPLC analysis of soil extracts demonstrated that both compounds disappeared more rapidly in the acid soil, and that the compounds disappeared at similar rates for both neutral soils, indicating that pH governed disappearance rates in these soils. Disappearance rates were generally first-order for both compounds, although NBPT disappeared at an accelerated rate at low concentrations, presumably due to its simultaneous conversion to BNPO. The effectiveness of both compounds in controlling urea hydrolysis was closely related to the concentrations of BNPO found in the soil. BNPO was generally maintained at higher concentrations following NBPT application than when BNPO was applied directly to soil.

Inhibition of acetylcholinesterase and acute toxicity of organophosphorous compounds to fish: a preliminary structure-activity analysis

The acute toxicity of twenty organophosphorous pesticides to guppies was determined and related to several physico-chemical properties by quantitative structure-activity relationships. Differences in LC 50 data were analysed in terms of: (a) differences in potencies to inhibit acetylcholinesterase, (b) differences in rates of activation into the oxon analogues, (c) differences in alkylating potencies and (d) differences in biotransformation and bioconcentration. The analysis showed that it was not possible to obtain a complete model of the LC 50 data in terms of the different processes. However, a QSAR analysis of the individual processes resulted in many interesting observations on the influence of the physico-chemical properties of the compounds on these processes.

Urease inhibitory activity associated with N-( n-butyl)thiophosphoric triamide is due to formation of its oxon analog

HPLC-purified N-( n-butyl)thiophosphoric triamide (NBPT) had no delectable urease inhibitory activity when tested with jack bean urease. However, considerable inhibition was associated with soil extracts when NBPT had been exposed in the soils for several hours. This activity was due to an NBPT derivative that was isolated by reverse phase HPLC. with NBPT and the derivative detected by a postcolumn system based on acid hydrolysis and phosphomolybdate complex formation. The purified derivative was shown to be highly active, giving 50% inhibition of jack bean urease at a concentration between 10 and 100 nM. Mass spectral analysis revealed the active derivative to be N-( n-butyl) phosphoric triamide. the oxon analog of NBPT.

Activation and degradation of the phosphorothionate insecticides parathion and EPN by rat brain

Cytochrome P-450-dependent monooxygenases are known to activate phosphorothionate insecticides to their oxon (phosphate) analogs by oxidative desulfuration. These activations produced potent anticholinesterases, decreasing the I 50 values to rat brain acetylcholinesterase almost 1000-fold (from the 10 −5 M range to the 10 −8 M range). Since the usual cause of death in mammals from organophosphorus insecticide poisoning is respiratory failure resulting, in part, from a failure of the respiratory control center of the brain, we investigated the ability of rat brain to activate and subsequently degrade two phosphorothionate insecticides, parathion (diethyl 4-nitrophenyl phosphorothioate) and EPN (ethyl 4-nitrophenyl phenylphosphonothioate). Microsomes from specific regions (cerebral cortex, corpus striatum, cerebellum, and medulla/pons) of the brains of male and female rats and from liver were incubated with the phosphorothionate and an NADPH-generating system. Oxon production was quantified indirectly by the amount of inhibition resulting in an exogenous source of acetylcholinesterase added to the incubation mixture as an oxon trap. The microsomal activation specific activity was low for brain when compared to liver [0.23 to 0.44 and 5.1 to 12.0 nmol · min −1 · (g tissue) −1 respectively]. The mitochondrial fraction of the brain possessed an activation activity for parathion similar to that of microsomes [about 0.35 nmol · min −1 · (g tissue) −1 for each fraction], but mitochondrial activity was slightly greater than microsomal activity for EPN activation [0.53 to 0.58 and 0.23 to 0.47 nmole · min −1 · (g tissue) −1]. Whole homogenates were tested for their ability to degrade paraoxon and EPN-oxon (ethyl 4-nitrophenyl phenylphosphonate), quantitated by 4-nitrophenol production. Specific activity for oxon degradation in liver was greater than that in brain [31 to 74 and 1.1 to 10.7 nmole · min −1 · (g tissue) −1 respectively]. Overall, the brain and liver had about 1.5- to 12-fold higher specific activities for degradation than activation depending on the compound used. These findings demonstrate that the brain possesses both phosphorothionate activation and oxon degradation abilities, both of which may be significant during exposures to organophosphorus insecticides.

Inhibition of trans-permethrin hydrolysis in Pseudoplusia includens (Walker) and use of inhibitors as dyrethroid synergists

A variety of organophosphorus compounds, carbamates, chelators, sulfhydryl group reagents, ions, and potential competitive inhibitors were tested as in vitro inhibitors of trans-permethrin hydrolysis by midguts of the soybean looper, Pseudoplusia includens (Walker). The nonpolar (aryl or long chain alkyl) organophosphorus compounds were found to be the most effective inhibitors, with some having I 50s of less than 10 −10 M. Paraoxon analogs with O-alkyl groups that were ethyl or longer were approximately equally effective inhibitors, while -thiono analogs were much less effective than -oxon analogs. For both carbamates and chelators the most effective compounds were those possessing aryl moieties. For sulfhydryl group reagents, both ions (Hg +2, Cu +2) and aryl compounds (γ-bromonitroacetophenone, 4-chloromercuriphenylsulfonic acid) were effective. Except for Hg +2, and Cu +2, the ions tested were relatively inactive. Several substituted 3-phenylthio-1,1,1-trifluoro-2-propanones produced I 50s of less than 10 −7 M. The oxidative inhibitors, pyrethroids, and other esters, amides, and alcohols examined were only moderately active (I 50 > 10 −6 M) at best. Of the compounds tested topically, the most potent synergists were the arylchloroorganophosphates, although a chelator (dibenzoylmethane) and a sulfhydryl group reagent (γ-bromonitroacetophenone), and 3-phenylthio-1,1,1-trifluoro-2-propanone showed some activity. The synergists that were effective for trans-permethrin on P. includens, were generally effective for fenvalerate on P. includens and trans-permethrin on Heliothis virescens (F.) as well.

Synergism of organophosphorus insecticides by diethyl maleate and related compounds in house flies

The toxicity of several organophosphorus and one carbamate insecticide for house flies is enhanced by simultaneous administration of diethyl maleate. Synergism factors vary from 2 to 116 and are strongly dependent on the combination of strain and insecticide studied. In general, it seems that thiono compounds are less synergized than their oxon analogs. Comparison of some analogs of maleic acid esters showed that the diethyl ester was not the most active compound. trans-Phenylbutenone was found to be a good alternative for diethyl maleate. Both diethyl maleate and trans-phenylbutenone deplete glutathione in the flies, but this depletion per se is certainly not the most important mode of action. In vitro experiments made clear that sufficient glutathione remained to permit a substantial insecticide degradation rate. Moreover, treatment of the flies with diethyl maleate 2–4 hr before the insecticide application resulted in a partial or complete disappearance of synergism in a period in which the glutathione concentration in the flies was still very low. Apart from this effect on the glutathione levels in the insect tissues it appeared from further experimental work in vitro that both compounds had a direct inhibitory effect on glutathione-dependent transferase(s) as well as on oxidative enzymes involved in insecticide degradation. The contribution of these reactions to the eventual synergism factor is discussed.

Comparative effects of organophosphorus insecticides on the activities of acetylcholinesterase, diacylglycerol kinase, and phosphatidylinositol phosphodiesterase in rat brain microsomes

The activities of acetylcholinesterase, diacylglycerol kinase, and phosphatidylinositol phosphodiesterase in rat brain microsomes were measured in the presence and absence of the organophosphorus insecticides, parathion and diazinon, and their respective oxon analogs, paraoxon and diazoxon. Marked inhibition of acetylcholinesterase (by 45–99%) was observed in the presence of paraoxon (10 −2–10 −6 M) and diazoxon (10 −2–10 −4 M). Reduction of acetylcholinesterase activity (by 22–33%) was achieved with the parent insecticides at high concentrations only (10 −2 M). In most cases, diacylglycerol kinase was insensitive to the pesticides. Marked stimulation of phosphatidylinositol phosphodiesterase (by 10–57%) was observed in the presence of all pesticides (10 −2–10 −3 M). The phosphodiesterase exhibited slightly greater sensitivity to the parent compounds compared to the oxon derivatives. Stimulation of the phosphodiesterase by the insecticides was not correlated with acetylcholinesterase inhibition. Accordingly, the increase in phosphodiesterase activity was judged not to be acetylcholine mediated, but rather represented a direct effect of the pesticides on the enzyme or its microenvironment. Based on the present in vitro observations, it is proposed that certain organophosphorus pesticides may interfere with the normal process of synaptic transmission through both the inhibition of acetylcholinesterase and the stimulation of phosphatidylinositol phosphodiesterase. In view of the high concentrations of pesticides required to elicit the latter effect, interpretation of its physiological significance must await results from further studies performed in vivo.

In vitro neurotoxic esterase assay using leptophos oxon analogs as inhibitors

In comparing neurotoxic esterase (NTE) inhibition properties of a series of phenylphosphonates, it was discovered that certain compounds including leptophos inhibited mipafox-insensitive phenylvalerate hydrolases. This leads to erroneous values for NTE inhibition which can be corrected by a differential assay: the total amount of mipafox-insensitive activity is determined with O-(2,6-dichlorophenyl) O-methyl phenylphosphonate and subtracted from the apparent NTE determined with the test compound before calculating pI 50's.