An insect acetylcholinesterase biosensor utilizing WO3/g-C3N4 nanocomposite modified pencil graphite electrode for phosmet detection in stored grains.

This study was undertaken to assess the potential of Tribolium castaneum (Red flour beetle) acetylcholinesterase (Tc-AChE) based electrochemical biosensor integrating WO3/g-C3N4 nanocomposite modified Pencil graphite electrode to detect an organophosphate insecticide, Phosmet. The WO3/g-C3N4 nanocomposite provides a non-toxic, biocompatible surface for binding the enzyme on the electrode surface, attributed to its large surface area, high conductivity, and low ohmic resistance. The proposed biosensor shows a very good analytical performance with LOD 3.6 nM for Phosmet and effectively determined Phosmet in wheat with a 99% recovery rate. Furthermore, molecular docking deciphers the binding interactions of Phosmet with Tc-AChE using a modified AutoDock LGA algorithm and an AMBER03 force field in YASARA. The kinetic parameters strongly suggest the high potency of inhibitor with the enzyme. This study presents an adaptable, rapid, and straightforward approach that opens ways towards real progress in developing commercial biosensors for pesticide detection.

Phosmet bioactivation by isoform-specific cytochrome P450s in human hepatic and gut samples and metabolic interaction with chlorpyrifos.

Data on the bioactivation of Phosmet (Pho), a phthalimide-derived organophosphate pesticide (OPT), to the neurotoxic metabolite Phosmet-oxon (PhOx) in human are not available. The characterization of the reaction in single human recombinant CYPs evidenced that the ranking of the intrinsic clearances was: 2C18>2C19>2B6>2C9>1A1>1A2>2D6>3A4>2A6. Considering the average human hepatic content, CYP2C19 contributed for the great majority (60%) at relevant exposure concentrations, while CYP2C9 (33%) and CYP3A4 (31%) were relevant at high substrate concentration. The dose-dependent role of the active isoforms was confirmed in human liver microsomes by using selective CYP inhibitors. This prominent role of CYP2C in oxon formation was not shared by other OPTs. The pre-systemic Pho bioactivation measured in human intestinal microsomes was relevant accounting for » of that measured in the liver showing two reaction phases catalysed by CYP2C and CYP3A4. Phosmet efficiently inhibited CPF bioactivation and detoxication, with Ki values (≈30 µM) relevant to pesticide concentrations achievable in the human liver, while the opposite is unlikely (Ki ≈ 160 µM) at the actual exposure levels, depending on the peculiar isoform-specific Pho bioactivation. Kinetic information in humans can support the development of quantitative in vitro/in vivo extrapolation and in silico models for risk assessment refinement for single and multiple pesticides.

Isolation of an isocarbophos-degrading strain of Arthrobacter sp. scl-2 and identification of the degradation pathway.

Isocarbophos is a widely used organophosphorus insecticide that has caused environmental pollution in many areas. However, degradation of isocarbophos by pure cultures has not been extensively studied, and the degradation pathway has not been determined. In this paper, a highly effective isocarbophos-degrading strain, scl-2, was isolated from isocarbophos-polluted soil. Strain scl-2 was preliminarily identified as Arthrobacter sp. based on its morphological, physiological, and biochemical properties, as well as 16S rDNA analysis. Strain scl-2 could utilize isocarbophos as its sole source of carbon and phosphorus for growth. One hundred mg/l isocarbophos could be degraded to a nondetectable level in 18 h by scl-2 in cell culture, and isofenphos-methyl, profenofos, and phosmet could also be degraded. During the degradation of isocarbophos, the metabolites isopropyl salicylate, salicylate, and gentisate were detected and identified based on MS/MS analysis and their retention times in HPLC. Transformation of gentisate to pyruvate and fumarate via maleylpyruvate and fumarylpyruvate was detected by assaying for the activities of gentisate 1,2- dioxygenase (GDO) and maleylpyruvate isomerase. Therefore, we have identified the degradation pathway of isocarbophos in Arthrobacter sp. scl-2 for the first time. This study highlights an important potential use of the strain scl-2 for the cleanup of environmental contamination by isocarbophos and presents a mechanism of isocarbophos metabolism.

Microbial degradation of phosmet on blueberry fruit and in aqueous systems by indigenous bacterial flora on lowbush blueberries (Vaccinium angustifolium).

Phosmet-adapted bacteria isolated from lowbush blueberries (Vaccinium angustifolium) were evaluated for their ability to degrade phosmet on blueberry fruit and in minimal salt solutions. Microbial metabolism of phosmet by isolates of Enterobacter agglomerans and Pseudomonas fluorescens resulted in significant reductions (P < 0.05; 33.8%) in phosmet residues on blueberry fruit. Degradation was accompanied by microbial proliferation of phosmet-adapted bacteria. Preferential utilization of phosmet as a carbon source was investigated in minimal salt solutions inoculated with either E. agglomerans or P. fluorescens and supplemented with phosmet or phosmet and glucose. Microbial degradation concurrent with the proliferation of P. fluorescens was similar in both liquid systems, indicative of preferential utilization of phosmet as an energy substrate. E. agglomerans exhibited the ability to degrade phosmet as a carbon source, yet in the presence of added glucose, phosmet degradation occurred within the 1st 24 h only followed by total population mortality resulting in no appreciable degradation. Characteristic utilization of glucose by this isolate suggests a possible switch in carbon substrate utilization away from phosmet, which resulted in toxicity from the remaining phosmet. Overall, microbial metabolism of phosmet as an energy source resulted in significant degradation of residues on blueberries and in minimal salt solutions. Thus, the role of adapted strains of E. agglomerans and P. fluorescens in degrading phosmet on blueberries represents an extensive plant-microorganism relationship, which is essential to determination of phosmet persistence under pre- and postharvest conditions.

Photodegradation of phosmet in wool wax models and on sheep wool: determination of wool wax bound phosmet by means of isotope ratio mass spectrometry.

The photochemical reactions of phosmet, an organophosphorus insecticide used for plant protection and for control of ectoparasites on productive livestock, were studied in the presence of wool wax. Induced by UV light, phosmet features numerous degradation pathways as well as photoaddition reactions with lipid structure moieties. In model irradiation experiments of phosmet in mixtures of solvents (cyclohexane, cyclohexene, 2-propanol) and fatty acid methyl esters (methyl stearate, methyl oleate, 12-hydroxymethyl stearate), both adjusted to the hydroxyl and iodine values of wool wax, half-lives were determined to be approximately 7 and 16 h, respectively. Irradiation of phosmet on crude sheep wool resulted in a degradation rate of 65% after 24 h. In tracer studies with stable isotope labeled phosmet ([15N]phosmet) in commercial lanolin and on raw sheep wool, employing a sunlight simulator and natural sunlight, wool wax bound phosmet was formed. After extraction and measurement by elemental analyzer/isotope ratio mass spectrometry, delta15N values of the phosmet-free wool wax fractions were notably increased as compared to the value of natural lanolin. Calculated from the delta15N values, an average of 13.9/15.6% (sunlight simulator/natural sunlight) was bound to wool wax lipids after irradiation of thin films of commercial lanolin. In experiments with sheep wool, 13.2 and 15.4%, respectively, were detected as wax-bound.

Sorption and degradation characteristics of phosmet in two contrasting Australian soils.

The organophosphate insecticide phosmet [phosphorodithioic acid, s-((1,3-dihydro-1,3-dioxo-2H-isoindol-2yl)methyl), o,o-dimethyl ester] is used to control red-legged earth mites (Halotydeus destructor), lucerne flea (Sminthurus viridis), and Oriental fruit moth (Cydia molesta) in horticulture and vegetable growing. This study was undertaken with two soils of contrasting properties to determine the extent to which sorption and degradation of the insecticide might influence its potential to leach from soil into receiving waters. Two soils were used: a highly organic, oxidic clay soil (Ferrosol) and a sandy soil low in organic matter (Podosol), sampled to 0.3 m depth. The extent of sorption and decomposition rate of a phosmet commercial formulation were measured in laboratory experiments. Sorption followed a Freundlich isotherm at all depths. The Freundlich coefficient K was significantly correlated (p = 0.005) with organic C content in the Podosol, and significantly correlated (p = 0.005) with organic C and clay content in the Ferrosol. K was highest (48.8 L kg-1) in the 0- to 0.05-m depth of the Ferrosol, but lowest (1.0 L kg-1) at this depth in the Podosol. Degradation followed first-order kinetics, with the phosmet half-life ranging from 14 h (0-0.05 m depth) to 187 h (0.2-0.3 m depth) in the Ferrosol. The half-life was much longer in the sandy Podosol, ranging from 462 to 866 h, and did not change significantly with depth. Soil organic C and to a lesser degree clay content influenced phosmet sorption and degradation, but the interaction was complex and possibly affected by co-solvents present in the commercial formulation.

A comparison of mutation spectra detected by the Escherichia coli lac(+) reversion assay and the Salmonella typhimurium his(+) reversion assay.

Each of the Escherichia coli tester strains in the WP3101P-WP3106P series contains an F' plasmid with a different base substitution mutation within the lacZ gene. Each of the six possible base substitution mutations, therefore, can be assayed with these strains by Lac(+) reversion. We used the strains to characterize the mutational profiles of 21 chemical mutagens, including alkylating agents, base analogs and oxidative compounds. We also assayed the mutagens with Salmonella typhimurium tester strains TA7002, TA7004 and TA7005, which detect A.T-->T.A, G.C-->A.T and G.C-->T.A mutations, respectively, and we compared the sensitivity and specificity of the two systems. Escherichia coli strain WP3102P was more sensitive than the S.TYPHIMURIUM: strains to G.C-->A.T transitions induced by N(4)-aminocytidine, 5-azacytidine, cumene hydroperoxide (CHP), t-butyl hydroperoxide (BHP), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methyl methane sulfonate and N-ethyl-N-nitrosourea (ENU), while the reverse was true for G.C-->A.T transitions induced by 2-aminopurine and phosmet. Escherichia coli strain WP3104P, which detects G.C-->T.A transversions, was superior to the S.TYPHIMURIUM: strains in detecting transversions induced by N(4)-aminocytidine, 5-azacytidine, 5-diazouracil, CHP, BHP, ENNG, ENU, 4-nitroquinoline 1-oxide (4-NQO) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Escherichia coli WP3105P was also more sensitive than S. TYPHIMURIUM: to A.T-->T.A transversions induced by N-methyl-N- nitrosourea (MNU), CHP and 4-NQO, but it was less sensitive to those induced by ENNG, ENU and 2-aminopurine. The present results indicate that the E.COLI: Lac(+) reversion system with tester strains WP3101P-WP3106P is as sensitive as the S.TYPHIMURIUM: His(+) reversion system for the detection of specific mutations induced by a variety of direct mutagens.

Phosphoinositide phosphorylation and shape changes produced by phosmet-oxon in human erythrocytes.

1. "In vitro" incubation of red blood cells with phosmetoxon induced crenated and invaginated forms. 2. [32P] phosphate incorporation was greater in membranes from erythrocytes exposed to 300 nM phosmetoxon for 10 min than in control cells. 3. The highest incorporation was for phosphatidylinositol (PI), followed by phosphatidylinositol phosphate (PIP) and phosphatidylinositolbiphosphate (PIP2). 4. An activation of phosphatidylinositol (PI) kinase was detected with 150 and 300 nM of the pesticide, while there was no change in poliphosphoinositides (PPI) phosphodiesterase activity. 5. Results suggest an association between changes in PI kinase activity, the phosphorylation cycle of phosphatidylinositols and alterations in erythrocyte morphology induced by phosmetoxon.

Comparison of measurement of dialkyl phosphates in milk/urine and blood cholinesterase and insecticide concentrations in goats exposed to the organophosphate insecticide, imidan.

Recognition of exposure to imidan was assessed in goats by dialkyl phosphate concentrations, blood cholinesterase (ChE) determinations, and blood imidan concentrations. Groups of three goats received 5.0 mg imidan/kg/day (low dose) or 10 mg imidan/kg/day (high dose) for 7 days orally. One goat received no imidan and one goat received an acute single dose (200 mg/kg). The urine of all treated goats was examined for the excretory dialkyl phosphates, O,O-dimethyl phosphorodithioate (DMDTP) and O,O-dimethyl phosphorothionate (DMTP). The overall mean DMDTP urinary concentration was 19.1 ppm (10-mg/kg treatment group) and 7.2 ppm (5-mg/kg treatment group). These metabolites rapidly disappeared following removal of the treatment except in those goats clinically affected. Milk contained no identifiable concentrations of dialkyl phosphates. Cholinesterase depression was observed in all imidan-treated goats, and a dose effect was observed. No imidan was detected in whole blood of either the 5- or 10-mg/kg treatment groups. Low blood concentrations (ppb) of imidan were measured in the acute single-dose exposed goat. Both urinary DMDTP and blood ChE provided recognition of imidan exposure. DMDTP, however, was immediately present in urine after exposure and provided stronger support for organophosphate exposure than did blood ChE.

[Effect of mixed function oxidases on phthalophos transformation in rat liver]. / Vliianie oksidaz smeshannoi funktsii na prevrashchenie ftalofosa v pecheni krys.

Phthalophos, organophosphoric pesticide, in the perfused isolated liver of rats transforms to water-soluble oxymethyl phthalimide and phthalimide. The induction of the liver hydroxylating enzymic system with milbex accelerates transformation of phthalophos: in 5 min only 14% of its initial amount is found in perfusate, while in the control--52%. The inhibition of the hydroxylating enzymic system with tetramethylthiuramdisulphide (dithiocarbamate pesticide) inhibits transformation of phthalophos: in 15 min perfusate contains 33% of its initial amount and the control--16%. Induction and inhibition of the hydroxylating enzymic system of the liver affect essentially the phthalophos toxicodynamics, decreasing the degree of the cholinesterase activity.

Metabolic and toxic behavior of phthalimide derivates. I. Fate of imidan in the fetus.

The organophosphate insecticide imidan includes a phthalimide group. Because of the structural relationship to the teratogenic compound thalidomide we have investigated its placental transfer and metabolic fate in rats. Its passage to embryonic tissues could be confirmed. After intraamniotic injection to the first amniotic cavity of the left uteri we could establish a distribution of imidan and 32P compounds throughout the uteri, the concentration depending upon the incubation time and the distances from the place of injection. The half-life of imidan, detected in the exterioized fetuses or in newborns, was 50--70 min. Imidoxon, i.e., the oxidized form of imidan, has a diminished half-life. This fact explicates the presence of imidoxon only in small concentrations after application of imidan.