Mechanism of the Selective Toxicity of Organophosphorus Compounds in the Armyworm, Leucania separata Walker. Port Title I. Topical Toxicity and Anticholincsterase Activity of Certain Organophosphorus Compounds SINCHAISRI, Neungpanich; MIYATA, Tadashi; SAITO, Author(s) Tetsuo Citation 防虫科学 (1977), 42(3): 125-132 Issue Date 1977-08-31 URL http://hdl.handle.net/2433/158971 Right Type Departmental Bulletin Paper Textversion publisher Kyoto University Mechanism of the Selective Toxicity of Organophosphorus Compounds in the Armyworm, Leueania eeparat« Walker. Part I. Topical Toxicity and Anticholinesterase Activity of Certain Organophosphorus Compounds. Neungpanich SINCHAISRI, Tadashi MIYATA and Tetsuo SAITO (Laboratory of Applied Entomology and Nematology, Faculty of A~riculture, Nagoya University, Chikusa·Ku, Furo-Cho, Nagoya, 461) Received May 9,1977. Boiyu-Kagaku, 42,125,1977. 19. 7? 3 '" .,1':13lj .QlrfJ I) :.t~$~J(1)iYf1Uljttl':OO9 .Qm~ I. tifllrfJ I) :.tl~S~J (1)l~m7Jt.::J I) :.t:r.~77 --tl~il~i'Fm '? 7';/lf:='":J '7- "'';/'7- .... 1 '" 1, trlIJiE. ~ijtkm::k (464ZtimI1Fffi1jrz~~1I1J ~tim*~Ml~$mEM~:tX3l) 52. 5. 9 ~Jm 4 f!l!:fHm ~ ~ ~Elt*,Jo.:> 7'7 ;l I-?, Leucania separate Walker .tIJEltII:;X",fi' ~ ~Elt:tJ ~ Il'Ombfliffl II:J; IJ WfjJ«~c c i,11: in vitro 1I::Io'1~ 7'7;l 1-?0.:>:1 ~ ~.x.;r.:r7--t! (ChE) mWf'Fffl~ HC-acetylcholine chloride ~mp"t"WfjJ«t=. LD,o filil;t. methyl parathion iJ{JR i, Ij, it <. "'?P'"'C' Ienltrothion, phenthoate, diazinon o.:>lIJii'"'C'ib -:>ti, 7'7;l I-? 0.:> ChE IHtfl ChE o.:>t,'Hl&~7f­ L tz; In vitro 11::1-3 It ~ ChE ~Il ;tHtm 1:1 phenthoate-oxon iJ{Ji"H ~ <, "'?P '"'C' methyl par­ aoxon, diazoxon, fenitroxon o.:>l!fl'i'"'C'ib -:> tz; 4 fiIi~rtU'i:Jo.:>~m:tJ c ChE m;tHtmo.:>IDMH~iHL t: c I:. -?, methyl parathion, diazinon, fenitrothion 0.:> 3 rn~m1fJ'"'C'o.:>mM l:tiI'!iiJ> -:>tz, -15, phenthoate o.:>.tJ~S methyl parathion 0.:> 5fffo.:>ChEmWf'Fm~7f-vt=iJ{~£!l:tJI;t. methyl parathion o.:>~ 1/3.8 i:ib-:>t=. UH,o.:>m1!l:1 phenthoate o.:>£!lU:J13"o.:>~~, ~ ~ PI;t.m14:J13'"'C'ft~l~o.:>~~iJ{ft!lo.:>3 fi11f1m~J c WI.. -:>"t"P ~ c ~'? 1iJfJlH't~IlIJ7f-v"t"P O. Armyworm, Leucania separata Walker is one Despite the measurement of ChE activity by the of the important pests of corn, Zea mays L. and method described Hestrln!" showed the unsatisf­ rice, Oryza sativa L.1,2> Many ecological and ied result in the preliminary procedure due to physiological works have been reported so far the strong interference of melanization effect in but it seems that publications concerning with the armyworm enzyme, the radioisotopic assay, the toxicological study on this species are still exercised in this study, offered much advantages lack. The toxicity test with 31 insecticides to and Quite desirable for the micro-volume of the armyworm larvae indicated that most of solution in the measurement of ChE activity. organophosphorus (OP) insecticides were highly Materials and Methods toxic whereas carbamates were not and endrin and dieldrin, the only two organochlorine com­ Insect pounds gave relatively high effects>. The colony of the armyworm larvae was obtain­ It is generally acknowledged that OP insecti­ ed from the University Farm, Nagoya University, cides kill animals by inhibiting cholinesterase Togo, Aichi prefecture in 1974. The larvae were (ChE). Most of them are rather poor inhibitors reared at 25'C in the plastic box, 24x30x5cm, of ChE in vitro. They owe their potency in vivo fed with fresh corn leaves as their basic food to the fact that they are converted in the body (partly fed with the leaves of Italian Rye grass to give compounds which are presumed to be to keep the successive generations during winter the direct inhibltorsv". season). The mass rearing technics were essen­ Thus, as the first approach to clarify the tially the same as the previous describeds.v, The mechanism of selective toxicity of four OP com­ 5th-instar, 2-3-day larvae were provided for pounds, methyl parathion, phenthoate, diazinon experiments. and fenitrothion against the armyworm, topical Chemicals toxicity of them and anticholinesterase activity Over' 975'6 purity of four organophosphorus of their oxygen analogs in vitro were investigated. compounds were used. Methyl parathion and 125 fenitrothion were received from Sumitomo Chem­ nerve cord (B & VNC), from the 5th-instar larvae ical Co., Ltd., phenthoate was obtained from of the armyworm were prepared separately. Nissan Chemical Industry Ltd., diazinon was ob Hornogenates of Hand WB were centrifuged at tained from Nihon Kagaku Co., Ltd. 900g for 5 min. The supernatants were used as Cholinesterase inhibitors, P=O analogs of these enzyme sources throughout the study. B & VNC 4 OP compounds were synthesized by an oxida­ homogenate was directly applied as the enzyme tion of P=S compounds in bromine solution and source without centrifugation. purified by thin layer chromatography (tic). Buffer's solution.-Phosphate buffer's solution, Acetyl [1- HC]-choline chloride (l4C-AChCl), 1/15M, pH 7.4, was mainly used in the routine 13.7 mCi/mmole, was purchased from Amersham works. In the experiment of an effect of pH on Co., England. The Amberlite CG-120, type 2, resin the ChE activity, buffer of Chadwick et al.8) was sodium salt was a product of Organo Co., Tokyo. used:NaCl, 26.30g; KH2PO" 3. SSg; NaOH, Chemicals for the preparation a scintillation 1.oog; water to make one litre. With all suspe­ solution, for instances, naphthalene (special grade), nsions, pH was adjusted to the desired value by 2,5-diphenyloxazole (PPO) , dimethyl POPOP and addtion of O.lN NaOH or O. IN HC!. The range l,4-dioxane were products of Katayama Chemical of pH values adjusted would cover enough in the ce., Ltd., Osaka. acidic and alkaline conditions from pH 6.1-9.2. Toxicity test Amberlite resin-dioxane mixture.-Preparation The evaluation of contact toxicity of 4 OP was essential the same as the method described compounds was accomplished by topical treating by Siakotos et al.w . Ten grams of dried resin the 5th-instar larvae of the armyworm with 0.7 was put into 100ml of dioxane. pi of acetone solution containing amount of OP Scintillation solution.-It was a modified Bray's compounds. In control, insects were treated with Cocktail!" containing 100g naphthalene,4g;PPO, the same volume of acetone. Insects were fed 200mg POPOP and 100ml absolute methanol, and with fresh corn leaves and kept in a constant then made to 1 litre with 1,4-dioxane. temperature chamber of 25·C. After 72hr, mor­ Assay procedure tality counts were taken. Badly affected or mori­ With an equal volume of 0.05ml, the mixture bund larvae were justified as dead insects accord­ of enzyme and HC-AChCl solution was put in a ing to the method of Ando and Sherman'!', At 20ml-tube, and the content of the tube was well least 4 replicates of 10 insects at 5-8 dosage mixed by shaking and incubated for 10 min at levels were provided for each insecticide in this 37·C. Non-enzymatic hydrolysis of the substrate test. The values of LD50 and LD 95 were calculated was measured with the solution containing of by the method of Finney12) and toxic index was 0.05ml of buffer instead of enzyme. ChE activity evaluated by the method of Sun!", was expressed as fl mole of the substrate hydro­ Radioisotopic assay of ekE activity lyzed/hr/g. Substrate solution.-Stock solution of l4C-AChCl, The proceeding steps of reaction were assayed 8 X1O-3M, containing approximate 3, 300, 000 as previously described by Siakotos et al.w . In cpm/ml was prepared by dissolving the contents order to stop the reaction, an approximately 5ml of AChCl .Irom a ampoule in distilled water. of the suspended resin-dioxane mixture was added. Stock solution of non-labelled AChCl, O. 55M, was Then, the enzyme-resin-dioxane solution was simultaneously prepared. The mixture of labelled brought to an exact volume of 10ml with dioxane. and non-labelled AChCl was provided to obtain The tube was tightly capped, and mixed by inver­ higher concentrations than 8 X 1O-3M of AChCl sion or shaking and allowed it to stand. The with highly enough radioactivity for the experi­ resin was allowed to settle by gravity for at ments. The mixture were always kept under least 2 hr. Five millilitres of the supernatant -20·C. solution was transferred to a scintilltion counting Source of enzyme--Three kinds of homogenate, vial, and 10ml of the modified Bray's scintilltion head (H), whole body (WB) and brain & ventral solution was added. Special care must be taken 126 to avoid transferring resin particles into the vial. HEAD BRAIN &VENTRAL' The samples, total volume of 15ml, were counted WHOLE BODY NERVE CORD. the radioactivity of the hydrolysis product in a U 10 100 U liquid scintillation spectrometer (Tricarb Model .<:: .<:: U U 3320 Liquid Scintillation Spectrometer) which < -e has an effeciency about 6O;'6at the optimized <3'" E HC setting. ::t. ....... 50 '-'~ For the inhibition study, inhibitors (P=O ~.E .- "- analogs of OP compounds) were dissolved in .~ "0 u .!::!'" acetone in widely different concentrations. With <3... w'" "0 the volume of O.Olml, an inhibitor solution was .<:: >. U .<:: delivered by means of a rnicrosyringe into the 20ml tube and allowed acetone to evaporate pS absolutely, 0.05ml of enzyme solution, thenafter, Substrate was added and pre-Incubated at 37°C for 30 min Fig. 1. Activlty-pS (-log mole of substrate) and then, 0.05ml of HC-substrate was added, curves for the enzymatic hydrolysis of incubated at 37°C for 20 min.