A comparison of cholinesterase activity after intravenous, oral or dermal administration of methyl parathion

Time-dependent changes in blood cholinesterase activity caused by single intravenous, oral or dermal administration of methyl parathion to adult female rats were defined. Intravenous and oral administration of 2.5 mg/kg methyl parathion resulted in rapid (<60 min) decreases in cholinesterase activity which recovered fully in vivo within 30-48 h. In contrast, spontaneous reactivation of cholinesterase in vitro was complete within 6 h at 37 degrees C. Dermal administration of methyl parathion caused dose-dependent inhibition of cholinesterase activity which developed slowly (> or =6 h) and was prolonged (> or =48 h). Time- and route-dependent effects of methyl parathion on cholinesterase activity in brain and other tissues generally paralleled its effects on activity in blood. In conclusion, pharmacodynamics of methyl parathion differ substantially with route of exposure. Recovery of cholinesterase in vivo after intravenous or oral exposure may partially reflect spontaneous reactivation and suggests a rapid clearance of methyl parathion or its active metabolite methyl paraoxon. The more gradual and prolonged inhibition of cholinesterase caused by dermal administration is consistent with disposition of methyl parathion at a site from which it or methyl paraoxon is only slowly distributed. Thus, dermal exposure to methyl parathion may pose the greatest risk for long-term adverse effects.     

Parathion and methyl parathion toxicity and metabolism in piperonyl butoxide and diethyl maleate pretreated mice

Pretreatment of male mice with piperonyl butoxide, 400 mg/kg 1 h before challenge with insecticides, resulted in a 40-fold antagonism of the acute i.p. toxicity of methyl parathion but potentiated the toxicity of parathion two-fold. Piperonyl butoxide had no effect on the toxicity of the oxygen analogs of these insecticides, methyl paraoxon and paraoxon. Diethyl maleate (1 ml/kg) depleted liver glutathione by 80% after one hour, potentiated the toxicity of both methyl parathion and methyl paraoxon, and partially counteracted the protective effect of piperonyl butoxide on methyl parathion toxicity. Piperonyl butoxide delayed the onset of brain cholinesterase inhibition by parathion. Studies of the metabolism of the insecticides by liver homogenates in vitro demonstrated that piperonyl butoxide inhibited both the oxidative formation of the oxygen analogs (activation) and oxidative cleavage to p-nitrophenol and dialkylphosphorothioic acid (detoxification). While parathion metabolism was mostly oxidative, methyl parathion metabolism appeared to be predominantly via glutathione-dependent enzymes. Studies of in vitro distribution of the insecticides demonstrated that piperonyl butoxide pretreatment resulted in elevated tissue concentrations of parathion and methyl parathion; however, the rate constant for elimination from plasma for both insecticides was unaffected by piperonyl butoxide. The overall rate of metabolism of methyl parathion in vivo was approximately twice that of parathion. These results suggest that during piperonyl butoxide inhibition of oxidative activation and cleavage, methyl parathion detoxification continues through uninhibited glutathione-dependent pathways of metabolism. The net result is a reduction in the acute toxicity of methyl parathion. Lack of an effective alternate pathway of detoxification may explain the delayed but greater toxicity of parathion in piperonyl butoxide pretreated mice.     

Effects of single or repeated dermal exposure to methyl parathion on behavior and blood cholinesterase activity in rats

The effects of a single or repeated dermal administration of methyl parathion on motor function, learning and memory were investigated in adult female rats and correlated with blood cholinesterase activity. Exposure to a single dose of 50 mg/kg methyl parathion (75% of the dermal LD(50)) resulted in an 88% inhibition of blood cholinesterase activity and was associated with severe acute toxicity. Spontaneous locomotor activity and neuromuscular coordination were also depressed. Rats treated with a lower dose of methyl parathion, i.e. 6.25 or 12.5 mg/kg, displayed minimal signs of acute toxicity. Blood cholinesterase activity and motor function, however, were depressed initially but recovered fully within 1-3 weeks. There were no delayed effects of a single dose of methyl parathion on learning acquisition or memory as assessed by a step-down inhibitory avoidance learning task. Repeated treatment with 1 mg/kg/day methyl parathion resulted in a 50% inhibition of blood cholinesterase activity. A decrease in locomotor activity and impairment of memory were also observed after 28 days of repeated treatment. Thus, a single dermal exposure of rats to doses of methyl parathion which are lower than those that elicit acute toxicity can cause decrements in both cholinesterase activity and motor function which are reversible. In contrast, repeated low-dose dermal treatment results in a sustained inhibition of cholinesterase activity and impairment of both motor function and memory.     

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.     

Blood gene expression markers to detect and distinguish target organ toxicity

The purpose of this study was to investigate whether the expression of specific genes in peripheral blood can be used as surrogate marker(s) to detect and distinguish target organ toxicity induced by chemicals in rats. Rats were intraperitoneally administered a single, acute dose of a well-established hepatotoxic (acetaminophen) or a neurotoxic (methyl parathion) chemical. Administration of acetaminophen (AP) in the rats resulted in hepatotoxicity as evidenced from elevated blood transaminase activities. Similarly, administration of methyl parathion (MP) resulted in neurotoxicity in the rats as evidenced from the inhibition of acetyl cholinesterase activity in their blood. Administration of either chemical also resulted in mild hematotoxicity in the rats. Microarray analysis of the global gene expression profile of rat blood identified distinct gene expression markers capable of detecting and distinguishing hepatotoxicity and neurotoxicity induced by AP and MP, respectively. Differential expressions of the marker genes for hepatotoxicity and neurotoxicity were detectable in the blood earlier than the appearance of the commonly used clinical markers (serum transaminases and acetyl cholinesterase). The ability of the marker genes to detect hepatotoxicity and neurotoxicity was further confirmed using the blood samples of rats administered additional hepatotoxic (thioacetamide, dimethylnitrobenzene, and carbon tetrachloride) or neurotoxic (ethyl parathion and malathion) chemicals. In summary, our results demonstrated that blood gene expression markers can detect and distinguish target organ toxicity non-invasively.     

Studies of the enzymic mechanism of the metabolism of diethyl 4-nitrophenyl phosphorothionate (parathion) by rat liver microsomes

1. The metabolism of parathion by rat liver microsomes is affected by various enzyme inhibitors in a manner quite typical of the ;mixed-function oxidase' enzyme systems. 2. With many of these inhibitors (p-chloromercuribenzoate, Cu(2+), 8-hydroxyquinoline) the conversion of parathion into diethyl hydrogen phosphorothionate is less inhibited than conversion into diethyl 4-nitrophenyl phosphate (paraoxon). 3. Compounds containing reduced sulphur stimulate the overall metabolism of parathion. However, the conversion of parathion into diethyl hydrogen phosphorothionate is stimulated more than its conversion into paraoxon. 4. The metabolism of parathion to diethyl hydrogen phosphorothionate is also stimulated by EDTA, Ca(2+) and Ba(2+), but these stimulatory effects are not additive. 5. The electron acceptors FAD, riboflavine, menadione and methylene blue exhibit a concentration-dependent differential inhibition of the metabolism of parathion to diethyl hydrogen phosphorothionate and to paraoxon. 6. The concentration of parathion required for the half-maximal rate of production of diethyl hydrogen phosphorothionate is significantly different from the concentration required for half-maximal rate of production of paraoxon. 7. The results are discussed in terms of either two separate enzyme systems metabolizing parathion to diethyl hydrogen phosphorothionate and to paraoxon or two different binding sites for parathion, which share a common electron-transport pathway.     

Structure-activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta

The mechanism and substrate specificity of the phosphotriesterase from Pseudomonas diminuta have been examined. The enzyme hydrolyzes a large number of phosphotriester substrates in addition to paraoxon (diethyl p-nitrophenyl phosphate) and its thiophosphate analogue, parathion. The two ethyl groups in paraoxon can be changed to propyl and butyl groups, but the maximal velocity and Km values decrease substantially. The enzyme will not hydrolyze phosphomonoesters or -diesters. There is a linear correlation between enzymatic activity and the pKa of the phenolic leaving group for 16 paraoxon analogues. The beta value in the corresponding Br?nsted plot is -0.8. No effect on either Vmax or Vmax/Km is observed when sucrose is used to increase the relative solvent viscosity by 3-fold. These results are consistent with rate-limiting phosphorus-oxygen bond cleavage. A plot of log V versus pH for the hydrolysis of paraoxon shows one enzymatic group that must be unprotonated for activity with a pKa of 6.1. The deuterium isotope effect by D2O on Vmax and Vmax/Km is 2.4 and 1.2, respectively, and the proton inventory is linear, which indicates that only one proton is "in flight" during the transition state. The inhibition patterns by the products are consistent with a random kinetic mechanism.     

Studies on the metabolism of diethyl 4-nitrophenyl phosphorothionate (parathion) in vitro

1. The metabolism of the phosphorothionate parathion in vitro was examined by using [(32)P]parathion and microsomes isolated from the livers of various animal species. 2. The major metabolic products of parathion in this system in vitro were identified as diethyl 4-nitrophenyl phosphate (paraoxon), diethyl hydrogen phosphate, diethyl hydrogen phosphorothionate and p-nitrophenol. 3. The reaction leading to the formation of diethyl hydrogen phosphorothionate and p-nitrophenol requires the same cofactors (NADPH and oxygen) required for metabolism of parathion to its active anti-acetylcholinesterase paraoxon. 4. The enzyme activity towards parathion per unit weight of liver is increased some 65-130% by pretreatment of male rats with phenobarbital and 3,4-benzopyrene. 5. The metabolism of parathion is inhibited by incubation in a nitrogen atmosphere and in an atmosphere containing carbon monoxide. Pure oxygen is also inhibitory. These results are discussed in terms of a deficiency of oxygen for maximal activity as well as the lability of some component of the system to oxidation.     

Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases.

Paraoxon, the active metabolite of parathion, can be detoxified through a noncatalytic pathway by carboxylesterases and a catalytic pathway by calcium-dependent A-esterases, producing p-nitrophenol as a common metabolite. The detoxication patterns of carboxylesterases and A-esterases were investigated in vitro in the present study with a high tissue concentration (75 mg/mL rat liver homogenate or 50% rat serum solution) to more closely reflect enzyme concentrations in intact tissues. A final paraoxon concentration of 3.75 microM was used to incubate with liver homogenates or serum solutions for 5 seconds or 3, 5, 15, or 25 minutes; also 0.625, 1.25, 2.5, 3.125, 3.75, or 5.0 microM paraoxon (final concentration) was incubated with liver homogenates or serum solutions for 15 minutes. Phenyl saligenin cyclic phosphate and EDTA were used to inhibit carboxylesterases and A-esterases, respectively. Significant amounts of p-nitrophenol were generated with or without either inhibitor during a 15 minute incubation with paraoxon from low (0.625 microM) to high (5.0 microM) concentrations. The amount of p-nitrophenol generated via carboxylesterase phosphorylation was greater than via A-esterase-mediated hydrolysis in the initial period of incubation or when incubating with a low concentration of paraoxon. Plateau shape curves of p-nitrophenol concentration versus time or paraoxon concentration indicated that carboxylesterase phosphorylation was saturable. When incubated for long time intervals or with high concentrations of paraoxon, more p-nitrophenol was generated via A-esterase-mediated hydrolysis than from carboxylesterase phosphorylation. The ratio of paraoxon concentration to tissue amount used in in vitro assays of this study was equivalent to dosing a rat with toxicologically relevant dosages. These in vitro data suggest that both carboxylesterases and A-esterases detoxify paraoxon in vivo; carboxylesterases may be an important mode of paraoxon detoxication in initial exposures to paraoxon or parathion before they become saturated, whereas A-esterases may contribute to paraoxon detoxication in repeated exposures to paraoxon or parathion because they will not become inhibited and will remain catalytically active unlike the carboxylesterases. The importance of carboxylesterases in detoxication of paraoxon was verified by an in vivo study. In rats pretreated with tri-o-tolyl phosphate, an in vivo carboxylesterase inhibitor, brain acetylcholinesterase was significantly inhibited after intravenous exposure to parathion. No significant inhibition of brain acetylcholinesterase was observed in rats pretreated with corn oil.     

Cytotoxicity of organophosphate anticholinesterases

Summary Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neuroblastoma cells and hepatocytes, which was detectable by the Cytosensor? microphysiometer. The nerve gas ethyl-S-2-diisopropylaminoethyl methylphosphorothiolate (VX), at 10 μM, produced significant reduction in cell metabolism within 2 min, as measured by changes in the acidification rate of the medium. The reduction was dose-and time-dependent and irreversible after 4 h of exposure. Two alkaline degradation products of VX produced no cytotoxicity. Exposure for 24 h to 3 μM VX caused 36% and 94% irreversible loss of metabolism in hepatocytes and neuroblastoma cells, respectively. The insecticides parathion and chlorpyrifos stimulated hepatocyte metabolism but inhibited neuroblastoma cells. Their oxons were more active. Exposure of neuroblastoma cells for 4 h to VX, parathion, paraoxon, diisopropylfluorophosphate or chlorpyrifos gave an LC₅₀ of 65, 775, 640, 340, or 672 μM, respectively, whereas 24 h gave an LC₅₀ of 0.7, 3.7, 2.5, 29, and 31 μM, respectively. Preincubation of hepatocytes with phenobarbital enhanced their response to parathion and VX due to metabolic bioactivation. Atropine partially blocked the effects of VX and paraoxon on both cell types, which suggests the involvement of a muscarinic receptor as the target for cytotoxicity. There was no correlation between OP in vivo neurotoxicity and in vitro cytotoxicity. It is suggested that the former results from their cholinesterase inhibition, while the latter results from action on different targets and requires much higher concentrations.     

Modulation of the mutagenicity of heterocyclic amines by organophosphate insecticides and their metabolites

People are commonly exposed to organophosphorus ester (OP) insecticides through the treatment of pets, homes, lawns, gardens, workplaces and in commercial agriculture. Aromatic amines are another chemical class with wide human exposure particularly dietary heterocyclic aromatic amines (HAAs). Previously, we reported that specific aromatic amines and ethyl paraoxon (the metabolite of the insecticide ethyl parathion) induced enhanced mutagenic responses in Salmonella typhimurium. In the present study, we demonstrated that the mutagenicity of 2-acetoxyacetylaminofluorene (2AAAF) and the heterocyclic dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) was enhanced in the presence of the OP insecticides, ethyl parathion or methyl parathion or a metabolite (methyl paraoxon). The mutagenicity of 2-amino-3-methylimidazo-(4,5-f)quinoline (IQ) was increased by methyl parathion and methyl paraoxon but not by ethyl parathion. This mutagenic synergy was expressed in S. typhimurium strain YG1024. Mammalian microsomal activation was required for PhIP and IQ to express mutagenic synergy. Synergistic responses are rarely incorporated in risk assessment models, yet such responses are important in establishing accurate toxicological characteristics of agents. Under real world conditions where people are exposed to a multitude of agents, the results of this study raise a concern about the environmental and public health impacts of OP insecticides.     

THE INSECTICIDAL MATERIAL IN LEAVES OF PLANTS GROWING IN SOIL TREATED WITH PARATHION

After parathion solutions had been watered on to soil around the roots of cabbage plants with leaves infested with Brevicoryne brassicae, Myzus persicae or Pieris brassicae larvae, these leaves showed a toxic effect on the feeding insects. In comparable experiments, Aphis fabae on leaves of broad bean was not affected at dosages that damaged the plants. The effect on the cabbage plants was observed when the parathion was of the highest degree of purity, as shown by chemical tests and its negligible anticholinesterase activity, although it was greater with commercial grade material. It occurred when all possibility of a fumigant action was excluded.
When the roots of wheat plants were treated with solutions of pure parathion the leaf guttation fluid was toxic to Aëdes aegypti larvae and contained an active anticholinesterase. This was shown to be paraoxon; no parathion could be detected in the fluid. The paraoxon was formed rather slowly from the parathion when the roots and leaves of wheat seedlings were immersed in the solution but not in the presence of compost alone.
In plants treated with pure parathion the translocation of the paraoxon formed under the influence of the roots was sufficient to account for the toxic effects produced. With commercial parathion, analogues or isomers present as impurities may also be translocated. Both these sources of systemic poisons should be borne in mind when considering parathion treatment of greenhouse soil in which food crops are to be grown.     

Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.     

Role of genetic polymorphism of human plasma paraoxonase/arylesterase in hydrolysis of the insecticide metabolites chlorpyrifos oxon and paraoxon

Plasma paraoxonase is a polymorphic enzyme that hydrolyzes paraoxon, the neurotoxic, active metabolite of the insecticide parathion. This enzyme is specified by at least two alleles with frequencies of about .7 and .3 among Caucasoid populations. A specific assay was developed that measured the activity of human plasma paraoxonase without interference from serum albumin which contributes significantly to the hydrolytic breakdown of paraoxon at the high pH values used in many previous assays. There was an 11-fold variation in paraoxonase activities, and the population distribution was at least bimodal. However, this specific assay did not improve the discrimination between the three genetic classes: (1) homozygotes for the low-activity allele, (2) heterozygotes, and (3) homozygotes for the high-activity allele. Chlorpyrifos oxon--the neurotoxic metabolite of the organophosphorus insecticide chlorpyrifos (Dursban)--was hydrolyzed by the same plasma fraction that hydrolyzed paraoxon. There was only four- to fivefold variability in enzyme activity, and the population distribution was unimodal. Homozygotes for low paraoxonase activity ranged over almost the entire spectrum of chlorpyrifos oxonase activity. Possible differences in susceptibility to chlorpyrifos toxicity therefore are unlikely to be predicted by the paraoxonase genotype alone. The ratio of paraoxonase over that of chlorpyrifos oxonase provided an excellent method for genetic typing of the paraoxonase polymorphism, as did the substitution of phenylacetate for chlorpyrifos as the substrate.     

Purification and properties of the phosphotriesterase from Pseudomonas diminuta

The phosphotriesterase produced from the opd cistron of Pseudomonas diminuta was purified 1500-fold to homogeneity using a combination of gel filtration, ion exchange, hydrophobic, and dye matrix chromatographic steps. This is the first organophosphate triesterase or organophosphofluoridate hydrolyzing enzyme to be purified to homogeneity. The enzyme is a monomeric, spherical protein having a molecular weight of 39,000. A single zinc atom is bound to the enzyme and is required for catalytic activity. Incubation with metal chelating compounds, o-phenanthroline, EDTA, or 2,6-pyridine dicarboxylate inactivate the enzyme. The kinetic rate constants, kcat and kcat/Km, for the hydrolysis of paraoxon are 2100 s-1 and 4 x 10(7) M-1 s-1, respectively. The enzyme is inhibited competitively by dithiothreitol, dithioerithritol, and beta-mercaptoethanol. In addition to paraoxon the phosphotriesterase was found to hydrolyze the commonly used organophosphorus insecticides, dursban, parathion, coumaphos, diazinon, fensulfothion, methyl parathion, and cyanophos.     

Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k(cat)/K(m) value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

Altering the Substrate Specificity of Organophosphorus Hydrolase for Enhanced Hydrolysis of Chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k_cat/K_m value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.     

Mineralization of paraoxon and its use as a sole C and P source by a rationally designed catabolic pathway in Pseudomonas putida

Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into beta-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.     

Mutagenesis of organophosphorus hydrolase to enhance hydrolysis of the nerve agent VX

Organophosphorus hydrolase (OPH) is capable of hydrolyzing a wide variety of organophosphorus pesticides and chemical warfare agents. However, the hydrolytic activity of OPH against the warfare agent VX is less than 0.1% relative to its activity against parathion and paraoxon. Based on the crystal structure of OPH and the similarities it shares with acetylcholinesterase, eight OPH mutants were constructed with the goal of increasing OPH activity toward VX. The activities of crude extracts from these mutants were measured using VX, demeton-S methyl, diisopropylfluoro-phosphate, ethyl parathion, paraoxon, and EPN as substrates. One mutant (L136Y) displayed a 33% increase in the relative VX hydrolysis rate compared to wild type enzyme. The other seven mutations resulted in 55-76% decreases in the relative rates of VX hydrolysis. There was no apparent relationship between the hydrolysis rates of VX and the rates of the other organophosphorus compounds tested.