Jun-ichi Fukami

Insect glutathione S-transferase: Separation of transferases from fat bodies of American cockroaches active on organophosphorus triesters

Abstract Several glutathione S-transferases which catalyze the conjugation of reduced glutathione with organophosphorus triesters were separated from fat bodies of adult female American cockroaches, Periplaneta americana (L.). Two transferases (I, V) were active on diazinon and three transferases (II, III, IV) were active on methyl parathion. The transferase (I) active on the pyrimidinyl moiety of diazinon was distinguishable from the other transferases on the O-methyl portion of methyl parathion, as shown by chromatographic properties, and additionally it was almost inactive or less active on 3,4-dichloronitrobenzene, methyl iodide, p-nitrobenzyl chloride, trans-cinnamaldehyde, and 1,2-epoxy-3-(p-nitrophenoxy)propane. Transferase II had high activities with “aryl” and “aralkyl” compounds, transferase III with “epoxide” and “alkene,” and transferase IV with “alkyl,” “aryl,” and “aralkyl” compounds. This indicated that the transferases had overlapping substrate specificities. The molecular weight was 35,000–37,000 for both of the enzymes active on methyl parathion and diazinon. The pH optima with methyl parathion and diazinon were about 8.5 and 6.5, respectively. At a glutathione concentration of 5 mM, Michaelis constants were 0.28 and 0.13 mM for methyl parathion and diazinon, respectively.

Enzymatic conjugation of diazinon with glutathione in rat and American cockroach

Abstract The mechanism of cleavage of the pyrimidinyl-phosphate bond of 32 P- and pyrimidine-2- 14 C-labeled diazinon or 32 P-labeled diazoxon by soluble enzyme preparations from rat liver and fat body of American cockroach was studied. The reaction products were identified as diethyl phosphorothioic acid and S -(2-isopropyl-4-methyl-6-pyrimidinyl) glutathione, which were formed by conjugation of reduced glutathione and the pyrimidinyl moiety of diazinon with the simultaneous cleavage of the phosphate ester bond. Several tissues in cockroach and rat were active in this conjugation, but the highest activity was found in the fat body and the liver. The glutathione S -transferase catalyzing the conjugation was specific for glutathione, and could not be replaced by other SH compounds. Diazoxon, n -propyl, and isopropyl diazinons having the structure similar to diazinon were also cleaved to give the glutathione conjugates. The pH optimum was 6.5 for the fat body and 6.0 for the liver enzyme. Both enzymes were inhibited by various SH reagents, oxidized glutathione, and some chelating agents. The fat body enzyme showed marked sensitivity to inhibition by o -phenanthroline.

Enzymatic hydrolysis of diazoxon by rat tissue homogenates

Abstract The enzymatic hydrolysis of 32 P-labeled diazoxon was studied using tissue homogenates of rat and American cockroach. The order of the hydrolytic activities of rat tissues for diazoxon was as follows: liver > blood > lung > heart > kidney > brain. A liver enzyme hydrolyzing diazoxon to diethyl phosphoric acid and 2-isopropyl-4-methyl-6-hydroxypyrimidine was located in the microsomes. The activity of the microsomal enzyme was inhibited by EDTA, heavy and rare earth metal ions, and SH reagents. Ca 2+ activated the enzyme and protected it from inactivation. Mitochondrial and soluble enzymes from liver and a serum enzyme also hydrolyzed diazoxon and they were also activated by Ca 2+ . The removal of calcium bound to the microsomal enzyme protein by dialysis against EDTA led to a partially irreversible change of the enzyme. The hydrolysis of diazoxon by the Ca 2+ -requiring microsomal and serum enzymes was more rapid than that of paraoxon. Hydrolysis of diazoxon did not occur in American cockroach homogenates. This difference in the capacity to hydrolyze diazoxon between mammals and insects is discussed in relation to the selective toxicity of diazinon.

Oxidative metabolism of diazinon by microsomes from rat liver and cockroach fat body

Abstract Metabolism of 32 P-, ethyl-1- 14 C-, and pyrimidine-2- 14 C-labeled diazinon was studied using microsomal preparations from rat liver and American cockroach fat body. The oxidation of diazinon by both microsomal enzyme systems fortified with NADPH or NADH occurred through desulfuration, hydroxylation of the ring-alkyl side chain, and cleavage of the aryl phosphate bond. The major metabolic products of diazinon were hydroxydiazinon, diazoxon, and hydroxydiazoxon, which were all biologically active, and the others were identified as 2-isopropyl-4-methyl-6-hydroxypyrimidine, 2-(2′-hydroxy-2′-propyl)-4-methyl-6-hydroxypyrimidine, diethyl phosphorothioic acid, and diethyl phosphoric acid which were all produced by the cleavage of the aryl phosphate bond. The rat liver enzyme system showed a higher rate of the oxidative metabolism of diazinon than the American cockroach fat body system. EDTA stimulated the overall metabolism of diazinon. Especially the accumulation of diaxoxon from diazinon and that of hydroxydiazoxon from both diazoxon and hydroxydiazinon in the rat liver microsomal system were stimulated by EDTA. On the basis of these in vitro studies, the general pathways of oxidative metabolism of diazinon in the mammal and the insect were presented.

Inhibition of Chitin Synthesis by Diflubenzuron in Manduca Larvae

Diflubenzuron was found to inhibit cuticle production in final instar Manduca larvae when fed or applied topically. After topical application of 5 , ug diflubenzuron to the newly molted 5th instar larvae, the rate of cuticle deposition decreased to only two-thirds of normal thickness. In vitro it inhibited both endocuticle deposition and ecdysterone-initiated pupal cuticle synthesis by the epidermis. Both effects are due to its inhibition (1D50=1. 1 x 10-9 M) of glucose or glucosamine incorporation into chitin.

Effects of chlordimeform on rectus abdominis muscle of frog

Abstract The effects of chlordimeform on rectus abdominis muscle of frog were investigated. Chlordimeform (10 −3 M ) caused a slow contraction, and at lower concentration (10 −5 –10 −3 M ) it inhibited the acetylcholine-induced contraction in noncompetitive manner. When chlordimeform was applied to the muscle of Rana catesbiana , K + -induced contraction was also inhibited in noncompetitive manner. Whereas it had no effect on caffeine-induced contraction. Chlordimeform-induced contraction was not affected by respective addition of d -tubocurarine (10 −4 M ), procaine (10 −3 M ), or eserine (0.3 m M ), which results were same as that of K + -induced contraction. Chlordimeform, at lower concentration (10 −5 –10 −3 M ), inhibits the acetylcholine- and K + -induced contractions probably owing to depression of not only the sensitivity of endplate but also the excitability of cell membrane.

The in vitro metabolism of organophosphorus insecticides by tissue homogenates from mammal and insect

In Japan, dialkylaryl phosphorothioate insecticides such as ethyl parathion (O,O-diethyl O-p-nitrophenyl phosphorothioate), methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) and Sumithion [O,O-dimethyl O-(4-nitro-m-tolyl) — phosphorothioate] are widely used to control the larvae of the rice stem borer, Chilo suppressalis. Sumithion has very low toxicity against mammals (Nishezawa et al. 1961), although its chemical structure is very similar to that of methyl parathion which is highly toxic to mammals. Several years ago, we found the development of resistance for ethyl parathion to the larvae of rice stem borer. Therefore, it is worth while to study differences of metabolism of these insecticides in mammal, insect, and plant.

Effect of temperature on pyrethroid action to kdr-type house fly adults

Abstract The actions of DDT and some pyrethroids on the kdr -type 228e2b and susceptible SRS strains of Musca domestica L. were examined at five temperatures from 15 to 35°C. The two classes of insecticides showed a negative temperature coefficient of toxicity for both strains, although high resistance was detected over the temperature range examined. Penetration of (1RS)-trans -[ 14 C]-permethrin was positively correlated to temperature. Both permethrin and cypermethrin showed negative temperature coefficients on the exposed thoracic ganglia from the two strains. There was a slight difference in qualitative nerve response patterns of femur motor nerve preparations of the two strains to DDT and two types of pyrethroids applied to the exposed thoracic ganglia: The pattern of DDT is similar to that of pyrethroids with type I action rather than type II action. It is concluded that increased nerve sensitivity may be the reason for a negative temperature coefficient of pyrethroid toxicity to the two strains of house flies.

Metabolism of fenitrothion by organophosphorus-resistant and -susceptible house flies, Musca domestica L.

Abstract The metabolism of fenitrothion was investigated in highly resistant (Akita-f) and susceptible (SRS) strains of the house fly, Musca domestica L. The Akita-f strain was 3500 times more resistant to fenitrothion than the SRS strain. Fenitrothion, topically applied to the flies, was metabolized in vivo far faster in the Akita-f strain than in the SRS strain. In vitro studies revealed that fenitrothion was metabolized by a cytochrome P -450-dependent monooxygenase system and glutathione S -transferases. The former oxidase system metabolized fenitrothion in vitro into fenitrooxon and 3-methyl-4-nitrophenol as major metabolites, and into 3-hydroxymethyl-fenitrothion and 3-hydroxymethyl-fenitrooxon as minor metabolites. Glutathione S -transferases metabolized fenitrothion into desmethylfenitrothion. The cytochrome P -450-dependent monooxygenase system and glutathione S -transferases of the resistant Akita-f strain had 1.4 to 2.2 times and 9.7 times, respectively, as great activities as those of the susceptible SRS strain. These results suggest the importance of glutathione S -transferases in fenitrothion resistance in the Akita-f strain.

Linkage group analysis of nerve insensitivity in a pyrethroid-resistant strain of house fly

Abstract Nerve insensitivity was a major factor of mechanism of resistance to pyrethroids in the 228e2b strain of house fly. Reciprocal crosses between the resistant and susceptible SRS strains showed that resistance to permethrin was recessive in nerve sensitivity. Linkage group analysis by the F 1 male backcross method using multichromosomal marker strains was investigated electrophysiologically, following 10 −5 M permethrin application to the exposed thoracic ganglia of the backcross progenies. Results of the experiment showed that the recessive genetic factor responsible for nerve insensitivity to permethrin in the resistant strain is located only on the third chromosome.