STUDIES ON THE MECHANISM OF INSECTICIDAL ACTION OF ORGANO-PHOSPHORUS COMPOUNDS WITH PARTICULAR REFERENCE TO THEIR ANTIESTERASE ACTIVITY

The preparation of an extract of the mealworm larvae, Tenebrio molitor L. which hydrolyses ethyl butyrate and o -nitrophenyl acetate, but not acetylcholine, is described. The inhibition of this esterase by TEPP-containing materials and parathion was determined.
An enzyme that hydrolysed o -nitrophenyl acetate and was inhibited by a TEPP-containing material was demonstrated in the five other insect species used.
The relative toxicities as contact insecticides to adult Tribolium castaneum Hbst. of ten samples of TEPP-containing materials were compared with their relative activities as esterase inhibitors. There was not an exact quantitative correlation between TEPP content estimated chemically, insecticidal activity and anti-esterase activity; but the correlation was sufficient to suggest interdependence of these factors.
Eggs of Diataraxia oleracea L. and Ephestia kühniella Zell, were shown to contain an enzyme that hydrolysed o -nitrophenyl acetate and was inhibited by the TEPP-containing materials. This enzyme was present in eggs less than 24 hr. old, i.e. before there was any visible signs of development. The TEPP was shown to be toxic to these eggs and in high concentrations kills at an early stage of development before differentiation of the nervous system. This, in conjunction with the other evidence, suggests that esterases other than the choline-esterase of the nervous system are important when considering the toxic action of these compounds.
Comparison of the anti-esterase activity and toxicity of parathion and TEPP-containing materials as insecticides showed that although the TEPP materials were the more potent enzyme inhibitors, parathion was the more potent contact insecticide to five species of insects. This appears to be due to the relative instability of TEPP. The study of the rates of action of the two poisons applied at different concentrations supports this view.     

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.     

Saligenin Cyclic Alkyl Phosphates and Phosphorothionates with Insecticidal Activity

Several saligenin cyclic alkyl phosphates and phosphorothionates were prepared and their insecticidal activities were examined. 2-Methoxy-4H-1,3,2-benzodioxaphosphoran-2-one and its thiono analog are strong insecticides, the toxicities of which to a number of insects are almost equal to or superior to parathion. They are less toxic to mouse than parathion. When the length of alkyl radical was increased, the insecticidal activity decreased.     

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.     

A novel method for the assessment of methyl parathion by using coupling agents in environmental and commercial samples

A sensitive and rapid spectrophotometric method for the determination of reduced methyl parathion (=O,O-dimethyl O-(4-nitrophenyl) phosphorothioate) is described. The method is based on the interaction of diazotized reduced methyl parathion with 8-hydroxyquinoline (=quinolin-8-ol; 8-HQ) and 3-aminophenol (3-AP) as new coupling agents. Absorbances of the resulting chromophores are measured at 430 and 440 nm, respectively, and colored products were stable for at least 2 days. Beer's law is obeyed over the methyl parathion concentration range 0.2-5.5 microg ml(-1) for 8-HQ and 0.5-6.0 microg ml(-1) for 3-AP. From the data, it was confirmed that the two coupling agents can be effectively applied for the determination of methyl parathion in environmental and commercial samples.     

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.     

Mineralization of Parathion in the Rice Rhizosphere

We studied ¹⁴CO₂ evolution from ring-labeled [2,6-¹⁴C]parathion (O,O-diethyl-O-p-nitrophenyl phosphorothioate) in the rhizosphere of rice seedlings. The soil samples (nonflooded [60% water-holding capacity] and flooded) were treated first with technical parathion (20 μg/g) and then after 10 days with ring-labeled [¹⁴C]parathion. In unplanted soil, less than 5.5% of the ¹⁴C in the parathion was evolved as ¹⁴CO₂ in 15 days under both flooded and nonflooded conditions. In soil planted with rice, 9.2% of the radiocarbon was evolved as ¹⁴CO₂ under nonflooded conditions, and 22.6% was evolved under flooded conditions. These results suggest that soil planted with rice permits significant ring cleavage, especially under flooded conditions.     

Synthesis, X-ray structure, and anti-leukemic activity of oxovanadium(IV) complexes

In a systematic effort to identify and develop effective anticancer agents, four oxovanadium(IV) complexes with 1,10-phenanthroline (Phen) or 4,7-dimethyl-1,10-phenanthroline (Me2-Phen) as ligand(s) were synthesized and characterized. Among the four oxovanadium(IV) complexes synthesized, the crystal structure of the bis(phenanthroline)oxovanadium(IV) complex bis(1,10-phenanthroline)sulfatooxovanadium(IV) ([VO(SO4)(Phen)2], compound 1) has been determined. Compound 1 crystallized in the space group P2(1)/n with unit cell parameters a = 14.2125(17) A, b = 10.8628(13) A, c = 20.143(2) A, alpha = 90 degrees, beta = 102.569(2) degrees, gamma = 90 degrees, V = 3035.3(6) A3, and Z = 4. The refinement of compound 1 by full-matrix least-squares techniques gave an R factor of 0.0785 for 4356 independent reflections. The structure contains two enantiomorphous molecules, lambda and delta, which are related by an inversion center. Compound 1 exhibited 3.5-fold more potent cytotoxic activity against NALM-6 human leukemia cells than the mono(phenanthroline)oxovanadium(IV) complex (diaqua)(1,10-phenanthroline)sulfatooxovanadium(IV) ([VO(SO4)(Phen)(H2O)2], compound 2) (IC50 values: 0.97+/-0.10 microM versus 3.40+/-0.20 microM: P=0.0004). Methyl substitution in the phenanthroline ligand enhanced the anti-leukemic activity of the mono(phenanthroline)oxovanadium(IV) complex 4.4-fold (IC50 values: 0.78+/-0.10 microM, compound 4, versus 3.40+/-0.20 microM, compound 2; P=0.0003) and the anti-leukemic activity of the bis(phenanthroline)oxovanadium(IV) complex 5.7-fold (IC50 values: 0.17+/-0.02 microM, compound 3, versus 0.97+/-0.10 microM, compound 1; P=0.001). The leading oxovanadium compound, bis(4,7-dimethyl-1,10-phenanthroline)sulfatooxovanadium(IV) ([VO(SO4)(Me2-Phen)2], compound 3) triggered the production of reactive oxygen species (ROS) in human leukemia cells, caused G1-arrest and inhibited clonogenic growth at nanomolar concentrations.     

Activity of organophosphorus insecticides in bacterial tests for mutagenicity and DNA repair--direct alkylation versus metabolic activation and breakdown. II. O,O-dimethyl-O-(1,2-dibromo-2,2-dichloroethyl)-phosphate and two O-ether derivatives of trichlorfon

The following organophosphates were tested for their ability to induce DNA damage in a rec-type repair test with Proteus mirabilis strains PG713 (rec- hcr-) and PG273 (wild-type) and point mutations in the his- strain TA100 of Salmonella typhimurium: O,O-dimethyl-O-(1,2-dibromo-2,2-dichloroethyl)-phosphate (NALED); trichlorfon-O-methyl ether (TCP-O-ME), O,O-dimethyl-(1-methoxy-2,2,2-trichlorethyl)-phosphonate; trichlorfon-O-methyl ether vinyl derivative (TCP-O-MEVD), O,O-dimethyl-(1-methoxy-2,2-dichlorovinyl)-phosphonate. All compounds were negative in the repair test but induced base pair substitutions in S. typhimurium. The mutagenicity of NALED is due to the direct alkylating ability of the parental molecule and to mutagenic metabolites generated by enzymatic splitting of the side chain. Glutathion-dependent enzymes in the S9-mix eliminate the mutagenic activity of NALED completely. Mutation induction by TCP-O-ME and TCP-O-MEVD is predominantly caused by the reactive O-methyl ether configuration of the side chain and is resistant to metabolic inactivation by NADPH- or glutathion-dependent enzymatic pathways in the S9-mix of mice.     

Effect on Insecticidal Activity of Substituents at the 1,4-Benzodioxanyl Moiety of Haedoxan

The haedoxan analog, (±)-2-(2,6-dimethoxyphenoxy)-1-hydroxy-6-(6-methoxy-l,4-benzodioxan-7-yl)-3,7-dioxabicyclo[3.3.0]octane, and its congeners with 2-alkoxymethyl, 2-hydroxymethyl, 2-chloromethyl and 3-(3,4-methylenedioxyphenyl) substituents on the 1,4-benzodioxanyl group were synthesized from 6-methoxy-l,4-benzodioxan-7-carbaldehyde and its (±)-2- and 3-substituted derivatives, respectively. Some analogs were considerably insecticidal, although much less active than natural haedoxan A. The assay results suggest that 2,3-disubstitution on the 1,4-benzodioxanyl group was necessary to intensify the insecticidal activity.     

Linear alcohol ethoxylates: insecticidal and synergistic effects on German cockroaches (Blattodea: Blattellidae) and other insects

Sixteen linear ethoxylated alcohol surfactants (AEOs) were studied to determine their contact insecticidal activity to adult German cockroaches, Blattella germanica (L.) (Blattodea: Blattellidae). Within groups of AEOs of equal carbon chain length, insecticidal activity, measured as LT50 values (in minutes) and 24-h mortality after treatment, was inversely related to the amount of ethoxylation. There was a highly significant negative relationship between the hydrophile-lipophile balance (HLB) value of the AEO and contact toxicity. The AEO with the lowest HLB value, Tomadol 23-1 (HLB = 3.7), produced the greatest 24-h cockroach mortality. The contact activity of Tomadol 23-1 was evaluated against a wide range of other insect species. Most species were killed within 24 h by direct exposure (1-4 microl of a 50% ethanol solution) to Tomadol 23-1 or by spray exposure to an aqueous solution. Tomadol 23-1, at a sublethal concentration, was tested in combination with representative members of the carbamate, nicotinoid, organophosphate, pyrethrum, pyrethroid, and pyrrole insecticide classes. Significant synergism was demonstrated in combinations of Tomadol 23-1 and chlorfenapyr, clothianidin, imidacloprid, thiamethoxam, and pyrethrum. Tomadol 23-1 significantly reduced the insecticidal activity of propoxur, suggesting antagonism. The insecticidal activity of Tomadol 23-1 was synergized by coapplication with a sublethal amount of piperonyl butoxide, indicating the involvement of cytochrome P450 microsomal monooxygenases in insect metabolism of AEO surfactants.     

Insecticidal activity of inhibitors of polyamine biosynthesis on Spodoptera litura F. larvae.

The inhibitors of polyamine (PA) biosynthesis such as alpha-difluoromethylornithine (DFMO), methylglyoxal bis (guanylhydrazone) (MGBG) and bis (cyclohexylammonium) sulphate (BCHA) have been used to protect crop plants from pathogenic fungi. In this communication the insecticidal activity of these inhibitors on tobacco caterpillar, S. litura has been reported. All the inhibitors exhibited insecticidal activity; MGBG being more effective than others. The results suggest, for the first time, a possible avenue for the control of insect pests by specific inhibition of insect PA biosynthesis.     

POLYACRYLAMIDE GEL ELECTROPHORESIS OF RAT BRAIN ACETYLCHOLINESTERASE: ISOENZYME CHANGES FOLLOWING PARATHION POISONING1

Brain acetylcholinesterase (EC 3. 1. 1. 7) isoenzymes of 15- and 30-day-old rats were found to be inhibited by 2.5 mg/kg and 1.25 mg/kg dosage levels of intraperitoneally administered parathion (E-605; O, O-diethyl-p-nitrophenyl phosphorothionate). With 2.5 mg/kg dose level, the response of isoenzymes in 15- and 30-day-old rats was similar. At both ages, there was no significant sex difference in the degree of depression of the isoenzymes. There were no significant regional differences in the degree of inhibition of acetylcholinesterase isoenzymes in the rat brain. At 1.25 mg/kg dosage level, a differential isoenzyme inhibition was evident, with the major isoenzyme (isoenzyme 3) exhibiting the greatest sensitivity to the inhibitor in all brain areas examined. The course of isoenzyme depression and recovery following the administration of parathion differed in brain, serum and skeletal muscle. Whereas brain isoenzymes exhibited most marked inhibition at 2 h after injection, inhibition of serum and skeletal muscle isoenzymes was more prolonged. At 4 h after injection, these isoenzymes were still inhibited while brain isoenzymes had recovered to a substantial degree. Twenty four h following the injection of parathion, when brain and serum acetylcholinesterase isoenzymes had returned to control activity levels, isoenzymes of skeletal muscle demonstrated only minimal recovery.     

Deterrent and insecticidal chromenes and benzofurans from Encelia (asteraceae)

Four analogous chromenes and benzofurans that are among the major natural products of the genus Encelia were bioassayed for deterrency and toxicity against pest insects. The chromene encecalin has both antifeedant and insecticidal properties towards larvae of the noctuid pests Peridroma saucia and Plusia gamma. In addition to reducing larval growth and decreasing survivorship of neonate larvae fed artificial diets containing encecalin, this chromene was directly insecticidal to neonate larvae after 4 hr using a residue contact bioassay. Topical application of encecalin to third instar larvae of Peridroma led to a reduction in dietary utilization, indicative of a metabolic cost of handling the compound. The demethyl analogue of encecalin is considerably less active, and the benzofurans euparin and methyleuparin, all common constituents of Encelia, were essentially inactive or only weakly active in all bioassays against the insects.     

Synthesis and Biological Activity of O,O-Dialkyl S-(5-Aryl-l,3,4-oxadiazol-2(3H)-on-3-yl)methyl Phosphorothioates and Phosphorodithioates

Some phosphorus derivatives of oxadiazoles were synthesized to seek insecticidal lead compounds. The l,3,4-oxadiazol-2-ones were converted via the N-methylol derivatives to the corresponding N-chloromethyl derivatives. From these derivatives a variety of O,O-dimethyl phosphorodithioates 4, O,O-dimethyl phosphorothioates 5 and O,O-di-i-propyl phosphorothioates 6 were prepared.

These phosphorus derivatives were examined for insecticidal activity towards houseflies and for anti-acetylcholinesterase (anti-AChE) activity using the housefly heads as an enzyme source. Most of the compounds 4 and 5 showed contact toxicity as high as the analogous methidathion insecticides, which appeared to correlate with the strong anti-AChE activity. On the other hand, all the compounds 6 showed a high activity in AChE inhibition but only a poor insecticidal activity.     

Methyl parathion-induced sperm shape abnormalities in mouse.

Metacid 50, the commercial grade of methyl parathion (O,O-dimethyl-O-4-nitrophenyl phosphorothionate), a commonly used organophosphorus insecticide, was tested for its genotoxicity in Swiss albino mice using the sperm abnormality assay. Sperms of albino mice were examined at two time intervals, 1 week and 5 weeks after a single acute oral treatment with the pesticide at four dose levels, viz., 75.0, 37.5, 18.75 and 9.375 mg/kg body weight corresponding to 1/2 LD50, 1/4 LD50, 1/8 LD50 and 1/16 LD50 values respectively. A dose-related statistically significant increase in the percentage of abnormal sperm observed indicates the genotoxic potency of methyl parathion.     

A high-affinity ATP-binding site on 30S dynein

The enhancing effect of low concentrations (eg, 8 μM) of bis(4-fluoro-3-nitrophenyl)sulfone (FNS) on 30S dynein ATPase activity is increased when 1 mM dithiothreitol (DTT) is present. The effect of FNS + DTT is optimal at pH 7.5. Activation of the latent ATPase activity of 30S dynein by FNS + DTT is partially prevented by 1–3 μM ATP. Adenylylimidodiphosphate (AMP-PNP) is less effective than ATP, while β,γ-methylene-adenosine triphosphate (AMP-PCP), though a much stronger inhibitor of ATPase activity than AMP-PNP, does not protect against enhancement. These results demonstrate the presence of a high-affinity ATP-binding site on 30S dynein.     

Accelerated Mineralization of Two Organophosphate Insecticides in the Rhizosphere

Numerous xenobiotic compounds, including the organophosphate insecticides O, O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate (diazinon) and O, O-diethyl-O-p-nitrophenyl phosphorothioate (parathion), appear to be degraded in the soil environment by an initial cometabolic attack. Comparing the mineralization rates of radiolabeled diazinon and parathion in root-free and in rhizosphere soil, we tested our hypothesis that, because of the presence of root exudates, the rhizosphere is an especially favorable environment for such co-metabolic transformations. The insecticides were added individually at 5 μg/g to sealed flasks containing either soil permeated by the root system of a bush bean plant or identical soil without roots. Periodically, the flask atmospheres were flushed through traps and the evolved ¹⁴CO₂ was quantitated. Bush bean plant roots without associated rhizosphere microorganisms failed to produce a significant amount of ¹⁴CO₂. During 1 month of incubation, rhizosphere flasks mineralized 12.9 and 17.9% of the added diazinon and parathion radiocarbon, respectively, compared to 5.0 and 7.8% by the soil without roots. The mineralization of parathion but not of diazinon was stimulated in a similar manner when soil without roots was repeatedly irrigated with a root exudate produced in aseptic solution culture. Viable counts of microorganisms on soil extract agar were not significantly altered by root permeation or by root exudate treatment of the soil, leaving population selection and/or enhanced cometabolic activity as the most plausible interpretations for the observed stimulatory effects. Rhizosphere interactions may substantially shorten the predicted half-lives of some xenobiotic compounds in soil.     

Esterase polymorphism and sensitivity to dursban organophosphorus insecticide in Culex pipiens pipiens populations

Esterase polymorphism and Dursban (O,O-dimethyl-2-pyridylphosphorothioate) sensitivity have been investigated in 12 natural populations and three laboratory strains of Culex pipiens pipiens. This mosquito has two esterase loci, Est-1 and Est-2, which were shown to code esterases of the B group (aliesterases) but not cholinesterases. No correlation between Est-1 polymorphism and Dursban sensitivity was found, but the increase of the Est-2(0.64) allele in the populations less sensitive to Dursban was highly significant (r = -0.9850 for 6 df).     

Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies.