Naoki Motoyama

Interstrain comparison of glutathione-dependent reactions in susceptible and resistant houseflies

Abstract Glutathione S -alkyl- and S -aryltransferase activities and the glutathione-dependent reactions involved in the metabolism of diazinon, parathion, DDT and γ-BHC were determined in two susceptible and three resistant housefly strains. The relative rate of formation of desethyl diazinon and desethyl parathion and the degradation of γ-BHC paralleled the activities of the alkyl and aryltransferases in the various strains of houseflies suggesting that a single enzyme might be involved. DDT-dehydrochlorinase showed different relative rates among the strains indicating that the dechlorination was catalyzed by a different enzyme. The enzyme responsible for the conjugation of the pyrimidinyl moiety of diazinon appears to be different from the one which catalyzes the conjugation of the p -nitrophenyl moiety of parathion. The dearylation reactions were not mediated by the glutathione S -aryltransferase in the various housefly strains.

Purification and properties of housefly glutathione S-transferase

Abstract Glutathione S- transferases were partially purified from an insecticide resistant and a susceptible strain of houseflies and characterized using 3,4-dichloronitrobenzene (DCNB) as the substrate. The molecular weight of the enzyme was estimated to be 50,000 and SDS gel electrophoresis revealed that the enzyme consisted of two equal subunits of 23,000. An Arrhenius plot of temperature versus DCNB conjugation showed a discontinuity at about 35°C. The optimum pH for enzyme activity was 9.5 to 10. A difference in the equilibrium constants between the enzymes from the resistant and susceptible strains did not explain the higher overall reactions in the resistant strain. The same enzyme was active for methyl iodide conjugation, degradation of organophosphorus insecticides and γ-BHC, but was inactive for DDT-dehydrochlorination. The degradation of organophosphorus insecticides was via alkyl conjugation and/or “leaving group” conjugation.

Studies on the mechanism of azinphosmethyl resistance in the predaceous mite, Neoseiulus (T.) fallacis (family: Phytoseiidae)

An organophosphorus-resistant strain of the predaceous mite, Neoseiulus (Typhlodromus)fallacis (Garman), degraded more azinphosmethyl than a susceptible strain both in vivo and in vitro. The in vitro degradation of azinphosmethyl required glutathione as a cofactor, and the activity was associated with the soluble fraction of the mite. The major metabolite identified in vivo and in vitro was desmethyl azinphosmethyl. The higher rate of desmethylation of azinphosmethyl by the resistant mite appears to be responsible in part for resistance. No apparent difference in the pI50 was observed between the two strains in vitro, indicating that the mechanism of resistance was not associated with a modified cholinesterase. The resistant strain had a higher nonspecific estersase activity than the susceptible strain and also two extra electrophoretic esterase bands. These extra bands were also found with two other resistant strains.

in vitro metabolism of azinphosmethyl in susceptible and resistant houseflies

Abstract The in vitro metabolism of [ 14 C-methoxy] or [ 32 P]azinphosmethyl by subcellular fractions of abdomens from a resistant and a susceptible strain of houseflies was studied. The degradative activity in both strains was associated with the microsomal and soluble fractions and required NADPH and glutathione, respectively. The resistant strain possessed higher activity for both the mixed-function oxidases and the glutathione transferase than the susceptible strain, and both systems appear to be important in the resistance mechanism. The mixed-function oxidases were involved in the oxidative desulfuration as well as the dearylation of azinphosmethyl. A glutathione transferase located in the soluble fraction catalyzed the formation of desmethyl azinphosmethyl and methyl glutathione. This enzyme also demethylated azinphosmethyl oxygen analog. Although the soluble fraction exhibited both glutathione S -alkyltransferase and S -aryltransferase activity against noninsecticidal substrates, no evidence of the transfer of the benzazimide moiety from azinphosmethyl to glutathione was obtained. Sephadex G-100 chromatography of the soluble enzymes revealed a common eluting fraction responsible for both types of transferase activity.

Endogenous inhibitors of glutathione S-transferases in house flies☆

Abstract The inhibition of glutathione S-transferase by endogenous compounds present in the soluble fraction of house fly homogenates was investigated. The highest inhibition was found with the female abdomen and increased with incubation time and with an increase in the tissue concentration. The correlation of increased inhibition with a parallel increase in the darkening of the soluble fraction indicated a possible association with melanization, thereby suggesting quinones as the possible endogenous inhibitiors of glutathione transferase. In vitro experiments demonstrated that quinones produced by mushroom tyrosinase did indeed inhibit glutathione S-transferase. Inhibition by quinones can be prevented by including glutathione or bovine serum albumin in the homogenization buffer. The inhibitory activity of a variety of quinones and related compounds on purified glutathione S-transferase was investigated. Oxygenated aromatics with hydroxy groups in the 1,2- or 1,4-position or ketonic carbonyls in the 1,4-position are good inhibitors of glutathione S-transferase.

Genetic studies on glutathione-dependent reactions in resistant strains of the house fly, Musca domestica L

Abstract Genetic studies of glutathione-dependent reactions were conducted with a diazinon-resistant house fly strain in which resistance is controlled primarily by genes on chromsome II. The resistant strain was crossed with a susceptible strain which had mutant markers on chromosomes II, III, and V, and the F 1 was backcrossed to the susceptible strain. Glutathione transferase activities of the resultant eight phenotypes were measured using 3,4-dichloronitrobenzene, methyl iodide, and γ-benzene hexachloride as substrates. High levels of all these activities are controlled by gene(s) on chromosome II. Further analysis was made by introducing diazinon resistance into a susceptible strain via genetic crossing-over. Intermediate activity levels for 3,4-dichloronitrobenzene and methyl iodide conjugations were introduced along with intermediate levels of resistance. Assays of individual flies of the synthesized strain revealed they were heterogeneous for glutathione-dependent activities, consisting of individuals with low, intermediate, and high transferase activity. Based on these results, high levels of the glutathione-dependent enzymes are not a major biochemical mechanism responsible for diazinon resistance. It was also demonstrated that glutathione S -aryltransferase and S -alkyltransferase in the house fly, as measured with 3,4-dichloronitrobenzene and methyl iodide, are inseparable genetically and may, therefore, be the same enzyme.

Molecular weight, subunits, and multiple forms of glutathione S-transferase from the house fly

Abstract Glutathione S -transferase was purified 106 fold from an insecticide-resistant strain of the house fly. Polyacrylamide gel electrophoresis of the enzyme resolved multiple activity bands for 3,4-dichloronitrobenzene conjunction. Electrophoresis of the extracts of the individual bands suggested that they may be derived from the same enzyme. The molecular weight of the native enzyme and its primary dissociated form was estimated to be 54,000 and 37,000, respectively. The dissociated form showed a discontinuity at about 35 C in an Arrenhius plot, while the native enzyme exhibited a continuous line. SDS gel electrophoresis of the native enzyme and of the individual electrophoretic bands resulted in the separation of a predominant subunit with an approximate molecular weight of 22,700. Numerous minor bands with various molecular weights were also detected. It was suggested that the multiple forms of the house fly glutathione S -transferase observed in electrophoresis, or eluted from CM-cellulose columns, may be due to dissociation, aggregation, or binding with bromophenol blue which alters the molecular weight and the molecular charge of the enzyme.

Dual role of esterases in insecticide resistance in the green rice leafhopper

Abstract The role of esterases as related to insecticide resistance was studied in an organophosphorus (OP)-resistant strain of the green rice leafhopper. As judged by p -nitrophenyl acetate hydrolysis, 21, 5, and 74% of the esterase activity was located in nuclei/mitochondria, microsomes, and the soluble fraction, respectively. All the fractions were active in hydrolyzing malathion, paraoxon, and fenvalerate. Hydrolysis of malathion and fenvalerate increased with time while that of paraoxon reached a plateau within 15 min. Since a considerable amount of p -nitrophenol was detected in the paraoxon reaction at 0°C and at zero time, the formation of p -nitrophenol may be due to phosphorylation of the esterases rather than phosphorotriesterase action. The results suggest a dual role for esterases in resistance mechanisms; a catalyst for hydrolysis of malathion and fenvalerate, and a binding protein for the oxygen analogs of other OP insecticides, both of which would protect the intrinsic target, acetylcholinesterase, from inhibition. Chromatofocusing of the soluble fraction resolved five esterase peaks, I–V. These esterases were active toward the three general substrates as well as for the three insecticides tested, except for Peak I in which the overall activity was too low. Thin-layer agar gel electrophoresis showed that the chromatofocusing peaks I–V corresponded to the electrophoretic bands E 1 –E 5 , some of which were previously shown to be associated with OP resistance. The dual role of these esterases may explain the cross-resistance between malathion and other OP insecticides as well as synergism between OP and carbamate insecticides.

Multiple forms of esterases in mouse, rat, and rabbit liver, and their role in hydrolysis of organophosphorus and pyrethroid insecticides

Abstract Six to seven esterases from mouse, rat, and rabbit liver microsomes were resolved by chromatofocusing in the pH range 7–4. Each esterase peak showed a different substrate specificity pattern with the substrates evaluated. Malathion and paraoxon hydrolysis always corresponded with p -nitrophenyl acetate and methylthiobutyrate hydrolysis, whereas the pattern of fenvalerate hydrolysis was more complicated. Phosphorotriester hydrolase activity was isolated, and was found to be more specific toward paraoxon than toward the other insecticides. Time-course studies of paraoxon hydrolysis indicated that the hydrolysis of paraoxon by carboxylesterase was an inhibitory reaction. This reaction and phosphorotriester hydrolase activity can serve as a detoxication reaction toward organophosphate insecticides.

Studies on hydrolases in various house fly strains and their role in malathion resistance

Abstract Aliesterase, carboxylesterase, and phosphorotriester hydrolase activities in six house fly strains were studied in relation to malathion resistance. Selection of two susceptible strains with malathion for three generations resulted in an increase in both carboxylesterase activity and LD50 of malathion, indicating that the increased detoxication by the enzyme was the major mechanism selected for malathion resistance. With the highly resistant strains, however, the carboxylesterase activity alone was not sufficient to explain the resistance level, and the involvement of additional mechanisms, including phosphorotriester hydrolase activity, was suggested. The E1 strain, which had high phosphorotriester hydrolase activity but normal or low carboxylesterase activity, showed a moderate level, i.e., sevenfold resistance. Upon DEAE-cellulose chromatography, two or three esterase peaks were resolved from susceptible, moderately resistant, and highly resistant strains. The substrate specificity, the sensitivity to paraoxon inhibition, and the α β ratio of malathion hydrolysis were studied for each esterase peak from the different strains. The results suggested the existence of multiple forms of esterases with overlapping substrate specificity in the house fly.