Anticholinesterase activity of some major intermediates in carbacylamidophosphate synthesis: preparation, spectral characterization and inhibitory potency determination

Carbacylamidophosphates with the general formula RC(O)NHP(O)R1R2 constitute organophosphorus compounds that are used as insecticides, pesticides and ureas inhibitors. In this work, we studied the inhibition potency of CCl3-C(O)NHP(O)Cl21, CHCl2C(O)NHP(O)Cl(2)2, CH2ClC(O)NHP(O)Cl23 and CF3C(O)NHP(O)Cl(2)4, which are the major intermediates for carbacylamidophosphates synthesis towards human erythrocyte acetylcholinesterase (hAChe) activity using Ellman's modified kinetic method. Unexpectedly, it was observed that they were not only hydrolytically unstable but also inhibited hAChE in a similar manner to that produced by organophosphorus insecticides. Enzymatic data, bimolecular inhibition rate constants (ki) and IC50 values for inhibition of hAChE demonstrated that they are irreversible inhibitors and the inhibition potency of compound 2 (IC50 = 88 microM) was the greatest in comparison with compounds 1, 3 and 4. Also the electropositivity of the phosphorus atom and the hydrophobicity of the compounds demonstrated that these two factors play an additional effect and different role in the inhibitory activity of these compounds. Hydrolytic stability of the compounds was determined by 31P NMR monitoring of the loss of the parent molecules with D2O as a function of time. This study considers antiacetylcholinesterase activity according to the structural and the electronic aspects of compounds 1-4, according to IR, 1H, 13C and 31P NMR spectral data.     

Acetylcholinesterase inhibition by diaza- and dioxophosphole compounds: synthesis and determination of IC50 values

Cholinesterases are targets for organophosphorus compounds which are used as pesticides, insecticides, chemical warfare agents and drugs for the treatment of disease such as glaucoma or parasitic infections. Most organophosphorus compounds impart their toxic action via inhibition of cholinesterases by reacting at an essential serine hydroxyl group. The inhibition process depends on the leaving group, stereochemistry and reactivity of the organophosphorus compound. In this study, the inhibitory potency of two isoelectronic and isostructural diaza- and dioxophospholes A (CH3C6H3 O2P(O)Cl) and B (CH3C6H3(NH)2P(O)Cl) against human acetylcholinesterase (hAChE) was examined by spectrophotometric measurements based on Ellman's method. Results indicated that compounds A and B were irreversible inhibitors with IC50 values of 0.48 and 1.54mM, respectively and inactivation constants (k(i)) of 0.0363 and 0.0207min(-1), respectively. The differences in the inhibitory potency of two phosphole compounds is discussed with respect to their structures. In addition, the synthesis and characterization of compound A is discussed.     

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.     

Laboratory and field tests in 1966-67 on chemical control of wireworms (Agriotes spp.)

Of sixteen compounds applied to soil in laboratory tests, azinphos-ethyl, P2188 (O,O-diethyl S-chloromethyl phosphorothiolothionate), ‘Dursban’ (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate), P1973 (S-(N-methoxycarbonyl-N-methylcarbamoylmethyl) dimethyl phosphorothiolothionate), B77488 (O,O-diethylphosphorothioate O-esterwith phenylglyoxylonitrile oxime) and R42211 (O,O-diethyl O-(2-diethylamino-6-methyl-pyrimidin-4-yl) phosphorothioate) killed wireworms when first tested, but in second tests with the same soils only ‘Dursban’, P2188 and B77488 did so. Treating seeds with ‘Dyfonate’ (O-ethyl S-phenyl ethyl phosphonodithioate) or with ethion/γ-BHC mixtures killed few wireworms. Three field trials compared the organophosphorus insecticides ‘Dursban’, ‘Dyfonate’ and phorate with organochlorine standards. In trials with barley and potatoes the standard was 3 lb a.i./acre (3·36 kg/ha) of aldrin. The organophosphorus compounds increased plant stands of barley almost as much as aldrin, although they killed fewer wireworms; and they protected fewer potato tubers from wireworm damage. The third trial compared the organophosphorus compounds with 0·5 lb a.i./acre (0·56 kg/ha) γ-BHC sprayed on a site drilled with sugar beet seed dressed with dieldrin. The γ-BHC increased plant stands almost as much as did 3 lb a.i./acre of the organophosphorus insecticides, and killed as many wireworms.     

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.     

Nonlinear "hydrophobicity-antiesterase activity" model for various types of organophosphorous compounds

The dependence of antiesteratic activity on the structure of insecticides (RO)2P(O)SCH(COOEt)SP(O)(OR)2 (I) and (RO)2P(O)SCH(COOEt)OP(S)(OR)2 (II) was examined. Nonlinear regression equations (parabolic and bilinear) "hydrophobicity-antiesteratic activity" were derived. Basing on the studies of the relationships between hydrophobicity and individual constants, the detailed mechanisms were proposed for the interaction of type (I) and (II) compounds with the esterase active centers. The mechanisms implicate different kinds of sorbtion for compounds of type I and II. Applicability of bilinear models, similar to that of Kubinyi type, for analyzing the structure-antienzyme activity dependences was demonstrated. Thus, several equations were obtained starting from the literature data on inhibition of esterases with diverse organophosphorus compounds.     

Observations on Germination and Acetylcholinesterase Activity in Wheat Seeds

Wheat seeds treated with organophosphorus insecticides exhibitdelayed germination both in the laboratory and in the field.Since (i) organophosphorus compounds inhibit cholinesteraseenzymes in animals and (ii) acetylcholine and cholinesterasehave been reported to occur in some plants, the hypothesis waspropounded that organophosphorus insecticides inhibit cholinesteraseactivity during cereal seed germination. Using biochemical andphysiological techniques, this hypothesis was tested in thelaboratory on wheat seeds germinated with and without the organophosphorusinsecticide, chlorfenvinphos. Evidence is presented for in vitroactivity of acetylcholinesterase in wheat seedlings and inhibitionof this activity by the insecticide. The possibility is discussedof a link between delayed germination and anti-cholinesteraseactivity of organophosphorus insecticides Wheat seeds, Triticum aestwum L., acetylcholinesterase, electrophoresis, germination, assay     

Anticholinesterase activity of some major intermediates in carbacylamidophosphate synthesis: Preparation,spectral characterization and inhibitory potency determination

Carbacylamidophosphates with the general formula RC(O)NHP(O)R₁R₂ constitute organophosphorus compounds that are used as insecticides, pesticides and ureas inhibitors. In this work, we studied the inhibition potency of CCl₃C(O)NHP(O)Cl₂1, CHCl₂C(O)NHP(O)Cl₂2, CH₂ClC(O)NHP(O)Cl₂3 and CF₃C(O)NHP(O)Cl₂4, which are the major intermediates for carbacylamidophosphates synthesis towards human erythrocyte acetylcholinesterase (hAChe) activity using Ellman's modified kinetic method. Unexpectedly, it was observed that they were not only hydrolytically unstable but also inhibited hAChE in a similar manner to that produced by organophosphorus insecticides. Enzymatic data, bimolecular inhibition rate constants (k_i) and IC₅₀ values for inhibition of hAChE demonstrated that they are irreversible inhibitors and the inhibition potency of compound 2 (IC₅₀ = 88 μM) was the greatest in comparison with compounds 1, 3 and 4. Also the electropositivity of the phosphorus atom and the hydrophobicity of the compounds demonstrated that these two factors play an additional effect and different role in the inhibitory activity of these compounds. Hydrolytic stability of the compounds was determined by ³¹P NMR monitoring of the loss of the parent molecules with D₂O as a function of time. This study considers antiacetylcholinesterase activity according to the structural and the electronic aspects of compounds 1–4, according to IR, ¹H, ¹³C and ³¹P NMR spectral data.     

Interaction of insecticides with lipid membranes.

The permeability of liposome membranes is increased by organophosphorus and organochlorinated insecticides at concentrations of 10(-5)--10(-4) M. The order of effectiveness is similar to the toxicity of the compounds to mammals, and is the following for permeation of non-electrolytes and for valinomycin-induced permeation of K+: parathion greater than 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) approximately aldrin greater than malathion greater than lindane. The degree of effectiveness for X-537A-induced permeation of Ca2+ was the following: aldrin greater than or equal to DDT greater than parathion greater than malathion greater than lindane. The organophosphorus compound, ethyl azinphos (10(-4) M), dramatically increases the permeability of liposome membranes to all the tested substances, probably as a consequence of surfactant effects. Some organochlorinated insecticides appear to react with cation ionophores and modulate their motion across lipid membranes. It is suggested that the insecticides may exert some of their toxic actions by modifying certain mechanisms in the cell membrane.     

Inheritance of chorionic malformations and insecticide resistance by Spodoptera exigua

Abstract:  Effect of exposure to the organophosphorus insecticide – fenitrothion [O,O-dimethyl-O-(4-nitro-3-methyl)phenyl] – on four generations of Spodoptera exigua Hübner (Lep., Noctuidae) larvae, with regard to the mortality and structure of the chorion of eggs laid was tested. Generations varied in the total mortality rate, although all of them showed some survival rate. The third and the fourth generations showed the lowest mortality among tested ones. Exposure to fenitrothion caused malformations in eggs. The chorion revealed cracks and diminutions of the outer layer. The quality and quantity of malformations increased proportional to the duration of exposure, e.g. the second exposed generation reveald more prominent changes than the first one. Malformations were observed in the next generations, which were not exposed to pesticide. The follicular cells, which are responsible for the structure and sculpture of the eggshell, must have inherited the malforming mechanism. Most probably, malformation takes place during the late development of eggs in the ovarioles.     

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.     

两种寄生蜂对几种农药的敏感性测定

就平腹小蜂、荔枝蒂蛀蛾白茧蜂对几种常用农药的敏感性进行了测定.结果表明：生物性农药的印楝素、定虫隆、灭幼脲对2种寄生蜂均较安全;Bt对平腹小蜂的毒性较小;杀虫双对平腹小蜂的影响较小,但对蒂蛀蛾白茧蜂有较高毒性（2 h内死亡率70%）;拟除虫菊酯类农药对平腹小蜂有一定的毒性,而对蒂蛀蛾白茧蜂有很高的毒性（2 h内死亡率均为100%）;2种寄生蜂均对有机磷杀虫剂如敌百虫、辛硫磷、敌敌畏及氧乐果等极度敏感.表明蒂蛀蛾白茧蜂比平腹小蜂对化学杀虫剂更敏感.因此在综合防治中,尤其是当田间释放平腹小蜂时应合理选择使用杀虫剂,应以生物源性农药为首选,避免使用菊酯类农药,尤其要禁止使用有机磷类农药,以减少对平腹小蜂等寄生性天敌的杀伤.     

有机磷水解酶的挖掘、改造及应用

有机磷化合物是一类广泛用作杀虫剂、增塑剂、阻燃剂的有毒化学品,由于难以降解而在农产品、水体和土壤中逐渐累积,容易引发严重的食品安全和环境污染问题。有机磷的酶促降解具有反应速度高和绿色环保等优点,是当前的研究热点。本文综述了近年来在有机磷水解酶的挖掘、改造及应用方面的研究进展,提出了进一步发展所面临的挑战和未来的研究方向,旨在为有机磷化合物的生物降解研究提供参考。     

An investigation of the toxicity of insecticides to birds' eggs using the egg-injection technique

Twenty-five insecticides have been tested for their toxicity to hen embryos at various concentrations, using an egg-injection technique. Of the two major groups, the organophosphorus compounds are much more toxic than the organochlorine. Most organophosphorus compounds lower the hatch rate and cause teratogenic effects at 100 ppm. Most organochlorines do not harm the embryo at high dosages (up to 500 ppm), with notable exceptions to this among the cyclodienes. However, starvation of the hatched chicks suggests that the majority of organochlorine compounds can kill at this stage, though fed chicks survive. The solvent used affected toxicity; in general, insecticides are more toxic to the embryo when dissolved in corn oil than when dissolved in acetone. If these findings can be applied to wild birds, it can be assumed that the vast majority of insecticides are harmless to birds' eggs. They are either not toxic in the concentrations so far found in this country, or else unlikely to pass through the mother bird to the egg.     

In vitro human phase I metabolism of xenobiotics I: pesticides and related compounds used in agriculture and public health,May 2003

This is the first revision of a database covering human phase I enzymes and their isoforms that metabolize pesticides and related compounds. The original version included enzymes that metabolize chloroacetamide and triazine herbicides, and organophosphorus insecticides. This revision also includes carbamate, nicotinoid, and pyrethroid insecticides and insect repellents.     

Detection and selection of organophosphate and carbamate resistance in Culex quinquefasciatus from Saudi Arabia

Resistance to a number of organophosphorus insecticides and the carbamate propoxur has been detected in a field population of Culex quinquefasciatus Say from Jeddah, Saudi Arabia. Cross resistance studies indicate that the same mechanism(s) may produce resistance to the full range of these compounds.     

Bis-pyridiumaldoxime reactivators connected with CH2O(CH2)n OCH2 linkers between pyridinium rings and their reactivity against VX

New bis-pyridinium oxime reactivators connected with CH2O(CH2)n OCH2 linkers between two pyridinium rings were designed and synthesized, and their reactivation potency was evaluated for AChE inhibited by organophosphorus VX agent. Among the prepared compounds, 1,2-dimethoxy-ethylene-bis-N,N'-4-pyridiumaldoxime dichloride 5a was the most potent and appeared to be the most promising compound as a potential reactivator for AChE inhibited by organophosphorus VX agent.     

Interaction of insecticides with lipid membranes

The permeability of liposome membranes is increased by organophosphorus and organochlorinated insecticides at concentrations of 10⁻⁵−10⁻⁴ M. The order of effectiveness is similar to the toxicity of the compounds to mammals, and is the following for permeation of non-electrolytes and for valinomycin-induced permeation of K⁺: parathion > 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) ≈ aldrin malathion > lindane. The degree of effectiveness for X-537A-induced permeation of Ca²⁺ was the following: aldrin DDT > parathion malathion > lindane. The organophosphorus compound, ethyl azinphos (10⁻⁴ M), dramatically increases the permeability of liposome membranes to all the tested substances, probably as a consequence of surfactant effects. Some organochlorinated insecticides appear to react with cation ionophores and modulate their motion across lipid membranes.It is suggested that the insecticides may exert some of their toxic actions by modifying certain mechanisms in the cell membrane.     

INSECTICIDE RESISTANCE OF COTTON APHID IN NORTH CHINA

Abstract  The cotton aphid ( Aphis gossypii Glover) is one of the most important pests infesting cotton in the cotton areas of North China. Since 1953 organophosphorus insecticides such as parathion and systox have been used to control the aphids for keeping up good yield of cotton. After several years. the concentration and the amount of spray were increasing in the field. In the early 1980's highly effective pyrethroid insecticides such as decis and sumicidin were largely imported into China. When first used 2. 5% decis emulsion was diluted in the ratio from 1:10000 to 1: 12000. However in 1985 the resistance of cotton aphid to pyrethroids increased by 171 times in general, 3230 times in some cotton fields. Thus it has prompted us to investigate the mechanism of resistance to insecticides and to search for the strategy to control the resistant aphids.
Experiments showed that the use of synergists including SV₁ (O, O-diethyl, O-phenyl phos-phorothionate) and PB has given evidence indicating mixed function oxidases (MFO). α-NA esterases and α-NA carboxylesterase are involved in the formation of resistance. The results also showed that the sensitivity of AChE to paraoxon in resistant aphids was lower than that in susceptible aphids.
Experiments showed that SV₁ was particularly synergistic to organophosphorus or pyrethroid insecticides and had played an excellent role in overcoming the resistance of cotton aphids to insecticides.     

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.