Andrew J. Forgash

Nonoxidative enzymes in the metabolism of insecticides.

Two major classes of enzymes, i.e., hydrolases and transferases, comprise all the nonoxidative enzymes, and together these enzymes catalyze a wide variety of biotransformations of insecticides. The hydrolytic enzymes involved in insecticide metabolism are carboxylesterase (EC 3.1.1.1), arylesterase alkylamidase, and DFPase (EC 3.8.2.1). Recent experimental evidence suggests that carboxylesterase enzyme(s), formerly known to hydrolyze malathion-type insecticides, can also catalyze hydrolysis of a variety of diversified insecticidal esters such as benzilic acid derivatives, carbanilate compounds, and pyrethroids. These organophosphate-sensitive esterases, with the exception of the enzyme which hydrolyzes malathion, are all present in microsomes. Similarly, the action of amidases now extends to those insecticidal compounds of their intermediates which contain an aminoformyl (N-CHO) moiety. Arylesterase and DFPase catalyze the P-anhydride bond cleavage of the leaving group, a major hydrolytic pathyway for organophosphate insecticides. Transferal enzymes which are presently know to metabolize insecticidal organophosphates are GSH-S-alkyltransferase (EC 2.5.1.12) and GSH-S-aryltransferase (EC 2.5.1.13). These enzymes cleave P-O-R (R = alkyl) or P-0-X (X = aromatic), with subsequent transfer of the R or X group to glutathione. Regarding the other conjugating enzymes, UDP-glucuronyltransferase (EC 2.4.L.17), UDP-glucosyltransferase (EC 2.4.1.35), and arylamine acetyltransferase (EC 2.3.1.5), much work is needed to understand their interactions with insecticidal compounds. There is some evidence that arylsulfotransferase (EC 2.8.2.1) MAY PLAY A PROMINENT ROLE IN THE CONJUGATIVE MECHANISMS OF INSECTS.

The identification and distribution of the carboxylic esterases in the American cockroach, Periplaneta americana (L.)☆

Abstract No less than twelve separate esterolytic enzymes were disclosed by the electrophoretic analysis of the supernatant fraction from whole cockroach homogenates. These isolated enzymes exhibited definite substrate preferences, and the effect of various inhibitors indicated the presence of five different organophosphorus resistant esterases and six separate ali-esterases in addition to cholinesterase. The dissected tissues of the cockroach were assayed colorimetrically for esterase activity with alpha-naphthyl acetate as substrate. The nerve cord and the brain were the sites of greatest activity. The other tissues in the order of decreasing enzyme activity were: midgut, Malpighian tubules, fat body, foregut, hindgut, haemolymph, reproductive system, and muscle. The largest number and the major concentration of the organophosphorus resistant esterases were found in the alimentary tract and the haemolymph, while the most abundant number and amount of the aliesterases were observed in the nerve cord, brain, fat body, and integument. Cholinesterase appeared to be restricted to the nervous system. The results of injection studies revealed that TOCP is not a selective inhibitor for aliesterases in vivo .

Nadph oxidation by microsomal preparations of gypsy moth larval tissues

Abstract NADPH oxidation by microsomal preparations from several tissues of fifth-instar larvae of gypsy moth, Porthetria dispar Linn. was studied. The distribution of this activity was as follows: 53.9, 27.8, 9.6, 4.4, and 4.3 per cent in midgut, fat body, hindgut, Malpighian tubules, and foregut respectively. Relatively specific activities of these microsomal preparations were 100, 81, 77, 61, and 55 for hindgut, Malpighian tubules, midgut, foregut, and fat body. NADPH oxidation was rapid for the first 20 minutes of the reaction and exhibited a K m of 1.75 × 10 −5 M NADPH. Cytochrome C greatly enhanced NADPH oxidation without affecting the K m . Carbon monoxide inhibited microsomal oxidation of NADPH and the inhibition was partially reversed by cytochrome C. This suggested the possibility of the presence of cytochrome P 450 . SKF 525-A inhibited NADPH oxidation and the locus of inhibition appeared to be cytochrome P 450 . Sulphhydryl inhibitors, p -chloromercuri-benzoate, p -chloromercuriphenyl sulphonate and Cu 2+ , inhibited NADPH oxidation and the inhibition was not reversed by cytochrome C. This indicated the involvement of NADPH-cytochrome C-reductase in the electron transport chain in gypsy moth.

Drosophila melanogaster eggshell adhesive

Abstract Drosophila melanogaster eggshells contain an adhesive which cements the egg to fruit tissue. Similarity in staining of the female accessory glands and the adhesive layer on the eggshell, and lack of adhesive on mature oocytes dissected from the ovarioles, demonstrate that the adhesive is secreted by the accessory glands. Positive staining of the accessory glands with the periodic acid/Schiffs reaction and with protein stains, plus the identification of 8 to 10 amino acids and glucosamine in hydrolysed extracts of the egg adhesive, indicate that the adhesive may be classified as a mucoprotein.

The distribution and metabolism of fenvalerate in pyrethroid-resistant and susceptible house flies

Abstract The distribution and metabolism of radiolabeled fenvalerate were compared in pyrethroid resistant (R) and susceptible (S) house flies. At the S-LD30, there were significant differences between strains with regard to the penetration and distribution of fenvalerate and excretion of its metabolites. Penetration was faster is S resulting in two to three times higher internal levels of radiocarbon. Excretion of fenvalerate metabolites began 24 hr post-treatment in S. It began after 4 hr in R and continued in a steady state so that at 24 hr almost 50% of the topically applied dose had passed through the insect. Metabolism studies were conducted at doses (LD5) that affected but did not kill individuals of either strain. Using thin-layer chromatography and [14C]fenvalerate radiolabeled in either the alcohol or acid moiety of the molecule, seven metabolites were isolated and identified from the water-soluble internal fraction and the excrement in R and S. In addition, three alcohollabeled unknowns were isolated from acid-, glucosidase-, and sulfatase-treated fly excrement. Two of the unidentified metabolites were common to both strains of flies while one was isolated only from the acid-treated excrement of R. While there were no qualitative differences in identified metabolites between strains, there were quantitative differences. Since metabolism studies with R were done at a nine-fold higher treatment dose than S, R flies metabolized as much as eight times more fenvalerate than S. Piperonyl butoxide (PBO) was an effective synergist of fenvalerate in R flies. At least two times more internal fenvalerate and approximately two times less hydroxylated metabolites were detected in R flies treated with fenvalerate-PBO (1:1) than in R flies treated with an equivalent dose of fenvalerate alone. The effect of PBO on metabolism implicates a role for the mixed-function oxidase system in the resistance mechanism of this strain of house fly.

NADPH-cytochrome-c-reductase: changes in specific activity in gypsy moth larvae

Abstract NADPH-cytochrome- c -reductase from guts of gypsy moth, Porthetria dispar , oxidized NADPH at the following rates: 11·6, 41·9, and 57·6 nmol/30 min per 0·6 mg protein, in the third, fourth and fifth instar, respectively. The increase in specific activity may be due to a natural induction process controlled by materials in the food. This could also explain the observed decreases just prior to ecdysis at which time the larvae have ceased feeding.

Hydroxylation and demethylation by gut microsomes of gypsy moth larvae

Abstract 1. 1. Biotransformations of certain aromatic xenobiotics by microsomes from the gut of gypsy moth Porthctria dispar L. larvae were studied. 2. 2. The specific activities for hydroxylation (aniline), N-demethylation (N,N-dimethyl-p-nitrophenyl carbamate) and O-demethylation (p-nitroanisole) were 11.3, 8.0, and 7.0 nmoles product per hour, respectively. Bovine serum albumin enhanced these rates by 11.9, 50.9, and 24.5 per cent, indicating the presence of some inhibitory material in the microsomal preparation. 3. 3. Maximal N-demethylase activity was observed at 37°C., and at higher temperatures enzymic activity declined rapidly, probably through thermal deactivation. Highest activity was observed between pH 7.0 and 7.8. 4. 4. All three reactions were inhibited by mixed-function oxidase inhibitor, SKF525-A. The addition of Mg3+ did not improve enzymic activity.

Acrylamide gel electrophoresis of housefly esterases

Abstract The esterases in extracts of one susceptible and six insecticideresistant strains of houseflies, Musca domestica L., were studied by acrylamide-gel electrophoresis, using alpha-naphthyl acetate as substrate. The results show that: (1) esterase patterns varied considerably between strains and within strains; (2) the esterases, except for one, acted as anions under these conditions; (3) not all of the esterases were mobilized in these experiments; and (4) no clear-cut, consistent differences were observed between resistant and susceptible flies. The results are discussed in relation to the research performed on the same species with other separation media and in relation to the resistance patterns of the housefly strains.

DDT-dehydrochlorinase in the Mexican bean beetle, Epilachna varivestis Muls.☆

Abstract The DDT-dehydrochlorinase level throughout various stages of the beetle was determined and found to increase in proportion to the body weight except in the pupal stage. Here a 35 per cent reduction in enzyme activity was observed; however, following adult feeding the enzyme returned to its previous level. Log dosage-probit lines for topical applications of TDE were determined with adult beetles at various ages. The LD 50 s, expressed in μ g TDE per mg dry wt., showed a close correlation with the enzyme level, expressed in μ g TDE dehydrochlorinated per mg dry wt., except for the 3 days following eclosion. The adult tissues were dissected and assayed for enzyme activity. The reproductive organs possess 2·5 times the activity observed in the second most active tissue, the alimentary canal. The other tissues in order of decreasing enzyme activity were: exoskeleton, flight muscles, central nervous system, and fat bodies.

Failure of Myo-Inositol to Prevent the Growth-Inhibiting Effects of Lindane in Periplaneta americana

Lindane, at a dietary concentration of 0.03%, retarded markedly the growth of the American cockroach ( Periplaneta americana (L.)) nymph, which requires an exogenous source of myoinositol. This effect is not permanent, however, for removal of the insects from the lindane-containing diet led to eventual recovery. Simultaneous addition of a large excess (6.6%) of myoinositol to the diet did not interfere with the action of the lindane. Delta BHC, at 0.133%, did not retard development.