Kenji Usui

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.

Hepatic disposition of parathion: Uptake by isolated hepatocytes and chromatographic translobular migration

Abstract Isolated rat hepatocytes suspended in Waymouths medium absorbed parathion rapidly and reversibly until the intracellular parathion concentration reached more than 300 times the ambient concentration. The distribution quotient q, defined as the ratio of intra- and extracellular concentrations at equilibrium, decreased when horse serum was added to the medium. The high hepatocyte affinity and rapid uptake of parathion suggested that this compound might be almost completely absorbed by periportal hepatocytes in the perfused liver and migrate downstream chromatographically through the lobule. Parathion infusion experiments verified this prediction and showed that the migration rate is dependent on the q value. The chromatographic feature may be useful as a basis for nonhistological investigation of intralobular hepatocyte heterogeneity. The lobule may function as a reverse-phase chromatograph also for many other unionized xenobiotics. Implications of the findings in the hepatic disposition of xenobiotics in vivo are discussed.

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.