Effect of Four Organophosphorus Insecticides on Microbial Activities in Soil

Laboratory tests were conducted with four organophosphorus insecticides, Bay 37289 (O-ethyl O-2,4,5-trichlorophenyl ethylphosphonothioate), diazinon [O,O-diethyl O-(2-isopropyl-4-methyl-6-pyrimidinyl) phosphorothioate], Dursban (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate), and Zinophos (O,O-diethyl O-2-pyrazinyl phosphorothioate), applied to a sandy loam at rates of 10 and 100 mug/g to determine whether these materials caused any serious effects on microbial activities related to soil fertility. All insecticides showed an effect on fungi and bacteria for the first and second week of incubation, but, subsequently, the populations returned to levels similar to those obtained in the controls. All insecticide applications increased ammonium production, but, in some instances, there appeared to be a slight depression of nitrification. Sulfur oxidation was equal to or better than that obtained with untreated soil in most cases. There was no significant effect on phosphorus mineralization. Oxygen consumption indicated that microbial respiration increased in proportion to the concentration of insecticides, suggesting the possibilities of microbial degradation of the insecticides or their degradation products and of uncoupling oxidative phosphorylation.     

Hapten synthesis for enzyme-linked immunoassay of the insecticide triazophos

Two haptens of the insecticide triazophos (O,O-diethyl O-[1-phenyl-1H-1,2,4-triazol-3-yl] phosphorothioate) were synthesized by introducing appropriate spacers in the O-ethyl site of the analyte molecular structure. First, thiophosphoryl chloride (PSCl(3)) reacts with methanol at low temperature to give O-ethyl dichlorothiophosphate. After reacting with 1-phenyl-3H-1,2,4-triazol, the O-ethyl dichlorothiophosphate was transformed into the intermediate O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) chlorothiophosphate. Then the intermediate reacts with 4-aminobutyric acid and 6-aminobutyric acid to produce hapten I and hapten II, respectively. The molecule structures of the two haptens were identified by (1)H nuclear magnetic resonance spectrum and mass spectrum. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was also developed to evaluate the two haptens. Results showed that the monoclonal antibodies with high titers were obtained after immunizing with protein conjugates of these haptens and that the immunoassay has high affinity and specificity to triazophos. These results suggested that the haptens were synthesized successfully and could be used for immunoassay for the rapid screening and sensitive determination of this insecticide.     

The Relationship Between Tobacco Yield and Time of Infection with Meloidogyne javanica

Yield of tobacco was related to the amount of infection by Meloidogyne javanica during the first month after transplanting. Six nematicidal treatments significantly reduced infection during this period and subsequently increased yield. However, during the second month after transplanting, infection in plots treated with O-ethyl S,S-dipropyl phosphorodithioate (V-C 9-104) and a mixture of 80% chlorinated C₃ hydrocarbons + 20% methyl isothioeyanate (DD + MENCS) was not significantly different from infection in untreated plots. After 3 months, root-knot indices in plots treated with V-C 9-104, DD + MENCS, O,O-diethyl O-[p-(methylsulfinyl) phenyl] phosphorothioate (B-25141), and 1,3-dichloropropene, 1,2-dichloropropane (DD) were not significantly different from those in untreated plots; reduced infection was present only in plots treated with ethylene dibromide (EDB) and 2-methyl-2 (methylthio) propionaldehyde O-(methylcarbamoyl) oxime (aldicarb). At the end of harvest (4 months after transplanting), root-knot indices in all plots were essentially equal.     

The Isomerization of Alkyl Phosphorothionates Induced by Carboxylic Acid Amides

Alkyl phosphorothionates are isomerized to phosphorothiolates by the catalytic action of dimethylformamide. Methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothionate) and sumithion (O,O-dimethyl O-3-methyl-4-nitrophenyl phosphorothionate) are more reactive than ethyl parathion (O,O-diethyl O-p-nitrophenyl phosphorothionate). Saligenin cyclic methyl phosphorothionate (salithion) decomposed to give a complicated pattern of products on thin layer chromatography. Besides S-methyl isomer, desmethyl sumithion (O-methyl O-3-methyl-4-nitrophenyl hydrogen phosphorothioate), 3-methyl-4-nitrophenol, methyl formate and dimethylamine were detected as reaction products from sumithion. Some other carboxylic amides including dimethylacetamide, acetamide and urea are also active. A reaction mechanism is proposed.     

Studies on Organophosphorus Insecticides

Various insecticides belonging to the O-alkyl-O-(subst. phenyl) phenylphosphonothioate family were prepared and their biological activities studied. Besides O-ethyl-O-(4-nitrophenyl) phenylphosphonothioate, which is utilized in these days, another compound, O-ethyl-O-(4-cyanophenyl) phenylphosphonothioate, proved to have excellent properties as an insecticide.

In our previous paper, it was reported that O,O-dimethyl-O-(4-cyanophenyl) phosphorothioate (S-4084) has only a low toxicity towards mammals though possessing a high activity towards rice stem borers. In this paper, the chemical and biological properties of S-4084 and its phenylphosphonothioate derivative (S-4087) will be reported in detail. It should especially be noted that S-4084 had a higher activity towards rice stem borers than S-4087 by topical or pot test in a room or in a green-house, but by field test S-4087 was stronger than S-4084.     

Nucleophilic N1-->N3 rearrangement of 5'-O-trityl-O2,3'-cycloanhydrothymidine

5'-O-Trityl-O2,3'-cycloanhydrothymidine (1) heated at 150 degrees C in the presence of O,O-diethyl phosphate or O,O-diethyl phosphorothioate anions undergoes rearrangement into N3-isomer (2); its structure was established by both advanced NMR methods and X-ray crystallographic studies. The most probable mechanism of 1-->2 rearrangement relies upon reversibility of glycosidic bond cleavage process.     

Metabolism of Cyanox®, O,O-Dimethyl O-(4-Cyanophenyl) Phosphorothioate in the Rats

Orally administered Cyanox, O, O-dimethyl O-(4-cyanophenyl) phosphorothioate, labelled with carbon-14 at p-cyano group was easily absorbed from the gastrointestinal tract of male Wistar rats, and distributed into tissues. Elimination of the radioactivity was rapid and essentially complete; namely during 96 hr approximately 90.7% and 10% of total radioactivity were excreted respectively into urine and feces. Carbon-14 in expiration was negligible. No detectable amount of Cyanox was found in the urine and 0.01% of the administered compound was present in the feces. Degradation products in the urine were identified as demethylcyanox, demethylcyanoxon, p-cyanophenol and p-cyanophenylsulfate.     

Transition-state structures for enzymatic and alkaline phosphotriester hydrolysis.

The primary and secondary 18O isotope effects for the alkaline (KOH) and enzymatic (phosphotriesterase) hydrolysis of two phosphotriesters, O,O-diethyl p-nitrophenyl phosphate (I) and O,O-diethyl O-(4-carbamoylphenyl) phosphate (II), are consistent with an associative mechanism with significant changes in bond order to both the phosphoryl and phenolic leaving group oxygens in the transition state. The synthesis of [15N, phosphoryl-18O]-, [15N, phenolic-18O]-, and [15N]-O,O-diethyl p-nitrophenyl phosphate and O,O-diethyl O-(4-carbamoylphenyl)phosphate is described. The primary and secondary 18O isotope effects for the alkaline hydrolysis of compound I are 1.0060 and 1.0063 +/- 0.0001, whereas for compound II they are 1.027 +/- 0.002 and 1.025 +/- 0.002, respectively. These isotope effects are consistent with the rate-limiting addition of hydroxide and provide evidence for a SN2-like transition state with the absence of a stable phosphorane intermediate. For the enzymatic hydrolysis of compound I, the primary and secondary 18O isotope effects are very small, 1.0020 and 1.0021 +/- 0.0004, respectively, and indicate that the chemical step in the enzymatic mechanism is not rate-limiting. The 18O isotope effects for the enzymatic hydrolysis of compound II are 1.036 +/- 0.001 and 1.0181 +/- 0.0007, respectively, and are comparable in magnitude to the isotope effects for alkaline hydrolysis, suggesting that the chemical step is rate-limiting. The relative magnitude of the primary 18O isotope effects for the alkaline and enzymatic hydrolysis of compound II reflect a transition state that is more progressed for the enzymatic reaction.     

Nucleophilic N1 → N3 Rearrangement of 5′-O-Trityl-O2,3′-Cycloanhydrothymidine

Abstract

5′-O-Trityl-O²,3′-cycloanhydrothymidine (1) heated at 150°C in the presence of O,O-diethyl phosphate or O,O-diethyl phosphorothioate anions undergoes rearrangement into N³-isomer (2); its structure was established by both advanced NMR methods and X-ray crystallographic studies. The most probable mechanism of 1→2 rearrangement relies upon reversibility of glycosidic bond cleavage process.     

Induction of oxidative stress and histopathological changes by sub-chronic doses of triazophos

The effect of triazophos (O, O-diethyl O-1-phenyl-1 H-1, 2, 4-triazol-3-yl phosphorothioate), a widely used insecticide was studied on the induction of oxidative stress and histological alterations at sub-chronic doses in male albino rats. Oral administration of triazophos at concentrations of 1.64, 3.2 and 8.2 mg/kg body wt for 30 days produced dose as well as time-dependent increase in the lipid peroxidation (determined by malondialdehyde levels) and glutathione-S-transferase (GST) activity in serum with aconcomitant decrease in ferric reducing ability of plasma (FRAP) and blood glutathione (GSH) content. Histopathological examination of liver of triazophos-treated rats showed significant and progressive degenerative changes as compared to control, which could be due to induction of oxidative stress. However, no significant histopathological changes were observed in spleen, kidney and brain at either dose of triazophos with respect to control. These results indicated that oral administration of triazophos was associated with enhanced lipid peroxidation and compromised antioxidant defence in rats in dose and time-dependent manner. Thus the present study demonstrated for the first time the role of oxidative stress as the important mechanism involved in the stimulation of hepatic histoarchitectural alterations at sub-chronic doses of triazophos in rats.     

Metabolism of fensulfothion by a soil bacterium, Pseudomonas alcaligenes C1.

Fensulfothion (O,O-diethyl O-[4-(methylsulfinyl)phenyl]phosphorothioate), an organophosphorus pesticide used to control the golden nematode Heterodera rostochiensis, is used as a source of carbon by microorganisms isolated from soils treated with the pesticide. Two of the microbial isolates, Pseudomonas alcaligenes C1 and Alcaligenes sp. strain NC3, used more than 80% of the pesticide in 120 h in culture when supplemented as a source of carbon. P. alcaligenes C1, which showed maximal growth on fensulfothion, degraded the compound to p-methylsulfinyl phenol and diethyl phosphorothioic acid. The phenolic metabolite could be identified by conventional spectral analysis, whereas the spectral patterns of the phosphorus-containing metabolite suggested that the compound was complexed with some cellular molecules. However, utilization of the phosphoric acid ester and ethanol by P. alcaligenes C1 suggested that the microbe attacks fensulfothion by an initial hydrolysis of the compound and subsequent utilization of the phosphoric acid ester. The pathway of degradation of fensulfothion by P. alcaligenes is of great value in the detoxification of the pesticide residues and also in the environmentally stable phosphoric acid esters.     

Metabolism of fensulfothion by a soil bacterium, Pseudomonas alcaligenes C1

Fensulfothion (O,O-diethyl O-[4-(methylsulfinyl)phenyl]phosphorothioate), an organophosphorus pesticide used to control the golden nematode Heterodera rostochiensis, is used as a source of carbon by microorganisms isolated from soils treated with the pesticide. Two of the microbial isolates, Pseudomonas alcaligenes C1 and Alcaligenes sp. strain NC3, used more than 80% of the pesticide in 120 h in culture when supplemented as a source of carbon. P. alcaligenes C1, which showed maximal growth on fensulfothion, degraded the compound to p-methylsulfinyl phenol and diethyl phosphorothioic acid. The phenolic metabolite could be identified by conventional spectral analysis, whereas the spectral patterns of the phosphorus-containing metabolite suggested that the compound was complexed with some cellular molecules. However, utilization of the phosphoric acid ester and ethanol by P. alcaligenes C1 suggested that the microbe attacks fensulfothion by an initial hydrolysis of the compound and subsequent utilization of the phosphoric acid ester. The pathway of degradation of fensulfothion by P. alcaligenes is of great value in the detoxification of the pesticide residues and also in the environmentally stable phosphoric acid esters.     

Involvement of two plasmids in fenitrothion degradation by Burkholderia sp. strain NF100

A bacterium capable of utilizing fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil. This bacterium was characterized taxonomically as being a member of the genus Burkholderia and was designated strain NF100. NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone. The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1.

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

Treatment with phosphodiester CpG-ODN ameliorates atopic dermatitis by enhancing TGF-β signaling

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG phosphorothioate (PS CpG-ODN) are known to decrease IgE synthesis in Th2 allergy responses. Nonetheless, the therapeutic role of PS CpG-ODN is limited due to cytotoxicity. Therefore, we developed a phosphodiester (PO) form of CpG-ODN (46O) with reduced toxicity but effective against allergies. In this study, we first compared the toxicity of 46O with CpG-ODNs containing a PS backbone (1826S). We also investigated the therapeutic efficacy and mechanism of 46O injected intravenously in a mouse model of ovalbumin (OVA)-induced atopic dermatitis (AD). To elucidate the mechanism of 46O underlying the inhibition of IgE production, IgE- and TGF-β-associated molecules were evaluated in CD40/IL-4- or LPS/IL-4-stimulated B cells. Our data showed that the treatment with 46O was associated with a lower hematological toxicity compared with 1826S. In addition, injection with 46O reduced erythema, epidermal thickness, and suppressed IgE and IL-4 synthesis in mice with OVA-induced AD. Additionally, 46O induced TGF-β production in LPS/IL-4-stimulated B cells via inhibition of Smad7, which suppressed IgE synthesis via interaction between Id2 and E2A. These findings suggest that enhanced TGF-β signaling is an effective treatment for IgE-mediated allergic conditions, and 46O may be safe and effective for treating allergic diseases such as AD and asthma.     

Separation of Lepidopterous Sex Pheromones by Reversed-phase Thin Layer Chromatography and High Performance Liquid Chromatography

Bis(4-chloro-2-ethylphenyl) phenylphosphonate was metabolically transformed into the cor-responding cyclic ester, i.e., 6-chloro-4-methyl-2-phenyl-4/f-1,3,2-benzodioxaphosphorin 2-oxide, in houseflies in vivo. In a p-unsubstituted analog, hydroxylation at the para-position of an ester linkage occurred preferably to alpha-hydroxylation with subsequent cyclization. The cyclization was diastereomerically selective, giving predominantly the cis ester. The biological activities of synthesized and related cyclic esters were similar to but weaker than saligenin cyclic phosphorus esters lacking a methyl group at the 4-position.     

Organophosphate toxicity in wild turkeys

An accidental poisoning of wild turkeys (Meleagris gallopavo silvestris) by O,O-Diethyl O-[p-(methylsulfinyl) phenyl] phosphorothioate is reported. Diagnosis was achieved by history, clinical observations, postmortem lesions, diagnostic therapy and pesticide analysis.     

Controlled release of DNA/polyamine complex by photoirradiation of a solid phase presenting o-nitrobenzyl ether tethered spermine or polyethyleneimine

Various gene transfer and automated/monitorized analytical applications require the controlled release of nucleic acid. A solid phase with spermine or polyethyleneimines (PEI, 600 MW) tethered by o-nitrobenzyl linkages was synthesized with polyethylene oxide beads (ArgoGel-NH(2)). The photolysis of test compound O-2-nitrophenethyl O,O-diethyl phosphate or solid phase with o-nitrobenzyl group as synthetic linker was completely degradable with photoirradiation at 365 nm for 10-18 min at 3.5 mW/cm(2). DNA binding with polyamine of the solid phase and releasing of DNA/polyamine were monitored by UV measurement and gel electrophoresis. The potential exists to employ a DNA-loaded solid phase for spatially, temporally, or dose-controlled release of DNA, at extracellular or intracellular sites.