Phosphorus-containing pesticide breakdown products: quantitative utilization as phosphorus sources by bacteria.

Bacteria were isolated that could utilize representatives of the following ionic phosphorus-containing breakdown products of organophosphorus pesticides as sole phosphorus sources: dialkyl phosphates, dialkyl phosphorothioates, dialkyl phosphorodithioates, alkyl arylphosphonates, alkyl arylphosphonothioates, and alkyl alkylphosphonates. Utilization of each organophosphorus compound, which was complete for 7 of 12 compounds studied, was confirmed by determination of protein yield from the amount of phosphorus source consumed. This is the first report of the utilization of an ionic dialkyl thiophosphate or dithiophosphate by microorganisms.     

The effects of thiophosphate substitutions on native siRNA gene silencing

RNA mediated interference has emerged as a powerful tool in controlling gene expression in mammalian cells. We investigated the gene silencing properties of six thiophosphate substituted siRNAs (all based on a commercial luciferase medium silencer) compared to that of unmodified siRNA. We also examined the cytotoxicity and dose-response using several thiophosphate modified siRNAs with unmodified siRNA. Our results show that two thiophosphate siRNA sequences convert from medium to high silencers with the addition of four randomly placed thiophosphates. Both thiophosphate siRNAs have a statistically significant difference in luciferase gene silencing (5% and 6% activity) relative to the unmodified native medium silencer referred to as siRNA-2 (18% activity) and four other thiophosphate siRNAs that maintain their medium silencing capability. This indicates that specific thiophosphate substitutions may alter native siRNA function. Further, this shows that thiophosphate siRNAs with the same nucleotide sequence but with different sulfur modification positions have different silencing effects. Both the native siRNA and the thio siRNAs showed a concentration dependent relationship, i.e., with concentration increase, the luciferase gene silencing effect also increased. Confirming cytotoxicity experiments showed no significant changes when HeLa cells were treated with 10nM thiophosphate siRNAs over the course of several days. These results suggest that specific placement of thiophosphates could play an important role in the development of siRNAs as therapeutics by engineering in properties such as strength of binding, nuclease sensitivity, and ultimately efficacy.     

The stereochemical course of D-glyceraldehyde-induced ATPase activity of glycerokinase from Escherichia coli

D-Glyceraldehyde-induced hydrolysis of adenosine (R)-5'-[gamma-17O,18O,thio]triphosphate catalysed by glycerokinase from Escherichia coli gives inorganic [16O,17O,18O]thiophosphate with the (S)-configuration, showing that the reaction proceeds with inversion of configuration at phosphorus. This result provides powerful support for the chemically most plausible mechanism, namely, that the hydrate of D-glyceraldehyde is the effective substrate which after phosphorylation or thiophosphorylation eliminates inorganic phosphate or inorganic thiophosphate, respectively, with regeneration of D-glyceraldehyde.     

Studies on the Organophosphorus Insecticides

During an investigation of organophosphorus insectiside, the present author found that the reactions of O,O-dialkyl phosphorochloridate or O,O-dialkyl phosphorochloridothioates with sodium salts of β-keto-esters or nitriles give new phosphoroates or phosphorothioates containg phenylacrylate or phenylacrylonitrile radicals, but do not give phosphonates.

The reactions of trialkylphosphites with α-halo- or α,α-dihalo-phenyl-β-keto-esters or nitrils also gave phosphoroates. Some phosphoroates prepared by this methods have high insecticidal activity.     

Evaluation of O,O-Dialkyl O-Cyanophenyl Phosphates and Phosphorothioates as Insecticides

Various O,O-dialkyl O-cyanophenyl phosphates and phosphorothioates were prepared and their biological activities were examined. Among them, O,O-dimethyl O- (4-chloro-2-cyanophenyl) phosphorothioate was found to have selective and high toxicity to houseflies. O,O-Dimethyl O- (4-cyanophenyl) phosphorothioate, O,O-diethyl O- (4-cyanophenyl) phosphorothioate and O,O-diethyl O- (2-chloro-4-cyanophenyl) phosphorothioate showed high insecticidal activty to American cockroaches, though the former two were not so effective to houseflies. The dimethyl esters of these series exhibited markedly lowered mammalian toxicity. Among the O-ethyl O-cyanophenyl phenylphosphonothioates, O-ethyl O- (2-chloro-4-cyanophenyl) phenylphosphonothioate was highly effective to mites, while less effective to insects.     

Degradation of methamidophos by <Emphasis Type="Italic">Hyphomicrobium</Emphasis> species MAP-1 and the biochemical degradation pathway

Methamidophos is one of the most widely used organophosphorus insecticides usually detectable in the environment. A facultative methylotroph, Hyphomicrobium sp. MAP-1, capable of high efficiently degrading methamidophos, was isolated from methamidophos-contaminated soil in China. It was found that the addition of methanol significantly promoted the growth of strain MAP-1 and enhanced its degradation of methamidophos. Further, this strain could utilize methamidophos as its sole carbon, nitrogen and phosphorus source for growth and could completely degrade 3,000 mg l⁻¹ methamidophos in 84 h under optimal conditions (pH 7.0, 30°C). The enzyme responsible for methamidophos degradation was mainly located on the cell inner membrane (90.4%). During methamidophos degradation, three metabolites were detected and identified based on tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC–MS) analysis. Using this information, a biochemical degradation pathway of methamidophos by Hyphomicrobium sp. MAP-1 was proposed for the first time. Methamidophos is first cleaved at the P–N bond to form O,S-dimethyl hydrogen thiophosphate and NH₃. Subsequently, O,S-dimethyl hydrogen thiophosphate is hydrolyzed at the P–O bond to release –OCH₃ and form S-methyl dihydrogen thiophosphate. O,S-dimethyl hydrogen thiophosphate can also be hydrolyzed at the P–S bond to release –SCH₃ and form methyl dihydrogen phosphate. Finally, S-methyl dihydrogen thiophosphate and methyl dihydrogen phosphate are likely transformed into phosphoric acid.     

Analysis of dialkyl phosphate metabolites in hair using gas chromatography-mass spectrometry: A biomarker of chronic exposure to organophosphate pesticides

The aim of our study was to develop and validate an analytical approach for the quantitative determination of three dialkyl phosphate (DAP) metabolites, dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP) and diethyl phosphate (DEP), of organophosphate pesticides (OPs) in hair samples. The proposed methodology comprises a decontamination step, solid–liquid extraction, followed by liquid–liquid extraction, pentafluorobenzyl bromide derivatization, clean-up on Florisil/PSA column and analysis by gas chromatography–mass spectrometry (GC-MS). Extraction recovery, obtained from 50?mg hair samples spiked at two concentration levels, ranged from 56.1 to 107.9% and the within-day precision ranged from 13.5 to 17.5%. Limits of detection (LODs) ranged from 0.02 to 0.10?ng mg^?1. The results obtained from the analysis of hair samples of 30 agricultural workers show the suitability of the proposed method for monitoring people occupationally exposed to OPs. The most frequently detected compound was DEP followed by DMP. This is the first report on the detection of dialkyl phosphates in human hair which reflects the ability of hair testing to assess chronic exposure to OPs.     

Acetylcholinesterase of cobra venom. Determination of concentration of active sites]

O-Nitrophenyl dimethylcarbamate and organophosphorus inhibitors O-n-propyl-p-nitrophenyl methylphosphonate, O-n-butyl-p-nitrophenyl methylphosphonate, O,O-diethyl-p-nitrophenyl phosphate, O-n-butyl-S-(beta-ethylmercaptoethyl) methylthiophosphonate, methysulphate of O,O-diethyl-S-(beta-phenyldimethylammoniumethly) thiophosphate were used in the titration of acetylcholinesterase active site concentratration in Naja naja oxiana venom. No side reactions with the acetylcholinesterase molecule as well as with other components of the venom were observed. In titration the effective concentrations of organophosphorus inhibitors with asymmetric phosphorus were 50% of their analytical concentrations, since cobra venom cholinesterase showed practically absolute stereoselectivity against the compounds.     

Studies on the Organophosphorus Insecticides

The reaction of O,O-dialkyl phosphorochloridates or phosphorochloridothioates with sodium salts of benzoylacetone and the reaction of trialkyl phosphites with α-chloro-benzoylacetone or acetophenone were attempted with a view to prepare the low toxic phosphorus insecticides containing acyl-vinyl group. It was found that the products did not have a phosphonate-form but a phosphorate-form and the phenyl-acyl-vinyl group had linkage with phosphorus atom by the enol-form of next carbonyl group of the phenyl-group.     

Inorganic thiophosphate effects on chromaffin cell structure and function

The effect was determined of replacing medium inorganic phosphate with thiophosphate on the structure and function of cultured bovine chromaffin cells. Cell cultures were incubated in normal medium containing fetal bovine serum, phosphate free medium or similar medium supplemented with inorganic phosphate or thiophosphate. In contrast to the other media, cells cultured with thiophosphate medium for 3–4 days showed seriously compromised structure and functions. The cells lost 75% of their catecholamine content and their ability to secrete remaining catecholamines in response to nicotine stimulation. Radio-labelled thiophosphate was rapidly taken up by the cells and, in long-term experiments, was incorporated largely into a 97–121 kDa protein band on SDS-PAGE. Additional minor bands were found to a lesser, variable extent. Transmission electron micrographs of cells treated with thiophosphate showed extensive depletion of chromaffin vesicles and disruption of mitochondria, suggesting that the functional damage noted with these cells could be associated with damage to mitochondria. Analysis of general cell metabolic activity by conversion of the dye (3-[3,4-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide) to its formazan derivative indicated increased metabolic activity at early stages of exposure to thiophosphate followed by a decline with continued exposure, supporting the argument for an overall depression of cell metabolism. Uptake of the dye neutral red, which is avidly accumulated by chromaffin cells, was also reduced for cells exposed to thiophosphate. The data suggest that thiophosphate enters chromaffin cells and disrupts energy dependent cell functions, including catecholamine storage and secretion.     

Stereochemical course of thiophosphoryl group transfer catalyzed by mitochondrial phosphoenolpyruvate carboxykinase

Guinea pig liver mitochondrial phosphoenolpyruvate carboxykinase catalyzes the conversion of (Rp)-guanosine 5'-(3-thio[3-18O]triphosphate) and oxalacetate to (Sp)-[18O] thiophosphoenolpyruvate , GDP, and CO2 by a mechanism that involves overall inversion in the configuration of the chiral [18O]thiophosphate group. This result is most consistent with a single displacement mechanism in which the [18O]thiophosphoryl group is transferred from (Rp)-guanosine 5'-(3-thio[3-18O]triphosphate) bound at the active site directly to enolpyruvate generated at the active site by the decarboxylation of oxalacetate. In particular, this result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.     

The stereochemical course of the ribosome-dependent GTPase reaction of elongation factor G from Escherichia coli

The stereochemical course of the ribosome-dependent GTPase reaction of elongation factor G from Escherichia coli has been determined. Guanosine 5'-(gamma-thio)triphosphate stereospecifically labeled with 17O and 18O in the gamma-position was hydrolyzed in the presence of the elongation factor and ribosomes. The configuration of the product, inorganic [16O, 17O, 18O]thiophosphate ws analyzed by 31P NMR after its stereospecific incorporation into adenosine 5'-(beta-thio)triphosphate. The analysis showed that the hydrolysis proceeds with inversion of configuration at the transferred phosphorus atom. It is therefore likely that the hydrolysis occurs in a single step by direct, in-line transfer of the phosphorus from GDP to a water oxygen, without a phosphoenzyme intermediate.     

The stereochemical course of phosphoric residue transfer during the myosin ATPase reaction

When adenosine 5'-(3-thiotriphosphate), stereospecifically labeled in the gamma position with 18O, was hydrolyzed in the presence of myosin subfragment 1 in 17O-enriched water, the product inorganic [16O,17O,18O]thiophosphate was chiral. The configuration of this product showed that the hydrolysis proceeds with inversion at the transferred phosphoric residue. This result suggests a direct, in-line hydrolysis mechanism for the ATPase.     

THE INSECTICIDAL ACTIVITY OF PARATHION, ITS ISOMERS AND SOME RELATED COMPOUNDS

Heat treatment of parathion at 140° C. and above resulted in isomerization and then thermal decomposition; the loss of toxicity to Calandra granaria being correlated with a reduction of the thiono-sulphur content. Similar results were obtained with O:O-dimethyl-(4-nitrophenyl)-thiophosphate. Paraoxon, O:S-diethyl-(4-nitrophenyl)-thiophosphate, and O:O-diethyl-S-(4-nitrophenyl)-thiophosphate, although all possessing considerable contact activity, were less insecticidal than parathion; O:O-dimethyl-(4-nitrophenyl)-thiophosphate, on the other hand, was considerably more effective than parathion. O:O- bis (2-chloroethyl)-O-(4-nitrophenyl)-thiophosphate was considerably less toxic than parathion and appeared to have a different mode of action. Compounds, S-ethyl- bis -(4-nitrophenyl)-thiophosphate, O-ethyl- bis -(4-nitrophenyl)-thiophosphate, and triethyl thiophosphate were ineffective as contact insecticides.
In the series of compounds examined, replacement of the group P = S by P = O or alteration in size of the groups attached to the central phosphorus atom caused a reduction of insecticidal activity.     

Determination of dialkyl phosphates in human hair for the biomonitoring of exposure to organophosphate pesticides

A new, simple, fast and sensitive method that enables the measurement of four dialkyl phosphates (DAPs) in human head hair is presented in the current study. The dialkyl phosphates, dimethyl phosphate (DMP), diethyl phosphate (DEP), diethyl thiophosphate (DETP) and diethyl dithiophosphate (DEDTP) are non-selective metabolites of the organophosphate pesticides (OPs). The extraction of DAPs from hair matrix was achieved by one step methanolic extraction. Head hair samples from general population and population occupationally exposed to OPs were analysed using gas chromatography–mass spectrometry (GC–MS) after derivatization with pentafluorobenzylbromide. The recovery of the target compounds was estimated at 84.3% for DMP, 116.1% for DEP, 109.0% for DETP and 91.5% for DEDTP. The limit of quantitation (LOQ) and detection (LOD) was 20 and 6 pg/mg for DMP, 10 and 5 pg/mg for DEP and DETP and 5 and 3 pg/mg for DEDTP, respectively. With-run and between-run precision as well as accuracy was estimated. The percentage of positive hair samples for DMP, DEP, DETP and DEDTP for the group of general population was 63.0%, 96.3%, 66.7%, and 70.4% respectively. The samples from the group with occupational exposure were positive for all dialkyl phosphates analysed. The median concentrations for DMP were 165.0 and 181.7 pg/mg, for DEP were 51.2 and 812.9 pg/mg, for DETP were 54.0 and 660.1 pg/mg, and for DEDTP were 40.0 and 60.6 pg/mg for the general population group and the group with occupational exposure respectively. Significant differences in the levels of the total dialkyl phosphates amongst exposed and not exposed groups were observed (p < 0.001). More specifically, the total ethyl phosphate (DEPs) and DAPs median concentrations were 119.5 and 301.5 pg/mg for the general population group and 1498.8 and 1694.4 pg/mg for the group with occupational exposure.     

Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate

In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled [gamma-17O,18O]ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[17O,18O]phosphate proceeds with inversion of configuration. Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb [Webb, M. R. (1982) Methods Enzymol. 87, 301-316]. We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O. These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate.     

The stereochemical course of phosphoric residue transfer catalyzed by beef heart mitochondrial ATPase

The stereochemical course of phosphoric residue transfer has been determined for beef heart mitochondrial ATPase. When aden 5'-(3-thiotriphosphate), stereospecifically labeled with 18O in the gamma position, was hydrolyzed in [17O]water in the presence of the ATPase, the product inorganic [16O, 17O, 18O]thiophosphate was chiral. The configuration of the product showed that the hydrolysis had proceeded with inversion at the gamma-phosphorus atom. This result suggests that there is a direct, in-line transfer of the phosphoric residue between ADP and water and that there is no phosphoenzyme intermediate.     

Structure-Activity Studies on Nematicidal Activity of Dialkyl Carbamates and Thiocarbamates

In laboratory tests, 129 dialkyl carbamates of types ROC(O)NHR'', RSC(O)NHR'', and ROC(S)NHR'' were tested in a screening bioassay against Panagrellus redivivus. The 10 most active were lethal at concentrations from 5 ppm down to ca. 1 ppm. Eight of these (the only ones active below 2.5 ppm) were thiolcarbamates (RSC(O)NHR''). Decyl N-methyhhiolcarbamate was also lethal to Meloidogyne incognita at approximately 1 ppm in direct contact tests.     

Influence of a Thiophosphate Linkage on the Duplex Stability - Does Sp Configuration Always Lead to Higher Stability Than Rp?

Abstract

ABSTRACT: In order to design an oligodeoxynucleoside phosphorothioate as an antisense molecule, it is important to establish the structure of the S-oligo with a strong affinity to the target RNA. In these molecules, internucleotide thiophosphate linkages produce diastereomers, the number of which increases in proportion to 2ⁿ (n: number of thiophosphate). To estimate the effect of this linkage on the duplex stability by UV melting curves, oligodeoxynucleotides having a single thiophosphate (referred to Soligo), dGCNsN'CG (s: thiophosphate, N, N′ = A or T), were prepared and their diastereomers isolated by HPLC. As demonstrated previously, the melting temperatures (Tm) for the Sp isomers were higher than those of the Rp when DNA was a target. On the other hand, it was found that for RNA as a target, the Rp isomers of dGCTsTCG and dGCAsTCG had higher stability than the Sp, and that the difference in the Tm values between the diastereomers was smaller than when DNA was a target. With dGCsTsACG, which has two thiophosphates, it was also found that the Tm values decreased with an increase in the number of thiophosphate linkages, and that the difference in Tm between the diastereomers was smaller when RNA was a target. Consequently, in practical clinical applications where RNA is a target, the influence of thiophosphate chirality on the duplex structure is almost negligible and Rp/Sp separation of an S-oligo may be of no major concern.     

Mevalonate-5-diphosphate decarboxylase: stereochemical course of ATP-dependent phosphorylation of mevalonate 5-diphosphate

Chicken liver mevalonate-5-diphosphate decarboxylase catalyzes the reaction of mevalonate 5-diphosphate (MVADP) with ATP to produce isopentenyl diphosphate, ADP, CO2, and inorganic phosphate. The overall reaction involves an anti elimination of the tertiary hydroxyl and carboxyl groups. To investigate the mechanism for transfer of the terminal phosphoryl group of ATP to the C-3 oxygen of MVADP, we have carried out the reaction using stereospecifically labeled (Sp)-adenosine 5'-O-(3-thio[3-17O2,18O]triphosphate) [( gamma-17O2,18O]ATP gamma S) in place of ATP. The configuration of the [17O,18O]thiophosphate produced was found to be Rp, corresponding to overall inversion of configuration at phosphorus in the thiophosphoryl group transfer step. This result is consistent with the direct transfer of the thiophosphoryl group from (Sp)-[gamma-17O2,18O]ATP gamma S to MVADP at the active site. Our result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.