W.C. Dauterman

Insecticide metabolism by multiple glutathione s-transferases in two strains of the house fly, Musca domestica (L)

Abstract Glutathione S-transferases from two strains of house fly have been prepared in a high degree of homogeneity by a procedure involving affinity chromatography and isoelectrofocusing. They fall into two groups in each strain. One group, of isoelectric point greater than pH 6.5, catalyzes the glutathione-dependent degradation of lindane, diazinon and methyl parathion. The other group, of low isoelectric point, has conjugating activity with the model substrate CDNB, but very little activity with the insecticide substrates. In the Cornell R strain the three isoenzyme forms in the high p I group appear to be almost identical in their substrate specificities. In the A strain, it is apparent that the enzyme forms falling into this group vary markedly in substrate specificity. The dehydrochlorination of DDT paralleled very closely the conjugation of the other insecticides catalyzed by the three high p I enzymes in the Cornell R strain. In the A strain, DDT dehydrochlorinase was most strongly associated with a glutathione S-transferase isoelectric at pH 7.1. It is tentatively concluded that multiple genes are involved in the production of the glutathione S-transferases involved in pesticide metabolism in the house fly and that DDT dehydrochlorinase may be derived from some, but not all, of these same genes.

Characterization of multiple glutathione transferases from the house fly, Musca domestica (L)

Abstract Glutathione transferases have been purified to a high degree of homogeneity from three strains of house fly by a procedure involving affinity chromatography on glutathione-sulfobromophthalein conjugate immobilized on Sepharose 4B, followed by preparative isoelectrofocusing. The affinity chromatography yielded purifications of between about 10- and 100-fold, depending on the strain and the substrate with which activity was measured. Each strain was shown to possess several proteins with glutathione S-transferase activity which fell into two clearly defined groups. The first group, of relatively low isoelectric point, showed activity with CDNB but little with DCNB, p-nitrobenzylchloride, or 1,2-epoxy-3-(p-nitrophenoxy)propane, whereas the second group, of higher isoelectric points, showed substantial activity with all substrates tested. Studies on the subunit structure of these enzymes demonstrated the existence of three different sized subunits of Mr 20,000, 22,000, and 23,500. From the experimental evidence recorded here, the existence of at least three functionally different glutathione transferases is inferred.

Induction of glutathione S-transferase by phenobarbital and pesticides in various house fly strains and its effect on toxicity

Abstract The effect of phenobarbital and certain pesticides on glutathione S-transferase activity was investigated. The maximum amount of enzyme induction occurred 96 hr after phenobarbital treatment. Chlorinated hydrocarbons were more effective inducers than the other pesticides evaluated. Phenobarbital treatment did not alter the apparent Km value but altered the V max value of glutathione S-transferase to 3,4-dichloronitrobenzene. The amount of reduced glutathione was not increased by phenobarbital treatment. Pretreatment of house flies with phenobarbital provides some protection against methyl parathion, methyl paraoxon, azinphosmethyl, and methidathion toxicity.

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.

The characterization by affinity chromatography of glutathione S-transferases from different strains of house fly

Abstract Glutathione S -transferases have been partially purified from five strains of house fly of defined resistance to insecticides. Apparent purification factors of 1.8–25 were obtained, the degree of purification depending on the assay substrate employed. Yields of up to 100% were obtained when 3,4-dichloronitrobenzene was used as substrate but with 1-chloro-2,4-dinitrobenzene as substrate the yields were of the order of 20–30%. Qualitative analysis of the proteins in these preparations indicated a high degree of similarity between the Cornell and Hirokawa strains and also a resemblance between the Fc and Rutgers strain. In the Cornell and Hirokawa strains, the bulk of the enzyme activity is attributed to two proteins (constituting a major fraction of the protein in the extracts) which appear to be of different isoelectric point from the proteins associated with enzyme activity in the Rutgers, Fc, and CSMA strains. A minor component in the Hirokawa and Cornell preparations appeared to correspond to the major catalytic protein in the Rutgers extract. On the basis of the above data, it is proposed that the various strains may possess a multiplicity of GSH S -transferases, common to all, individual proteins being present to varying extents in the different 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.

A comparative study of effects of atrazine on xenobiotic metabolizing enzymes in fish and insect, and of the invitro phase II atrazine metabolism in some fish, insects, mammals and one plant species

1. Atrazine (3 daily i.p. doses of 0.20 mg/kg or 10 ppb in the water for 14 days) did not change the xenobiotic metabolizing enzyme activities (XME) towards the substrates aldrin epoxidase (AE), NADPH-cytochrome c reductase (NCCR), 7-ethoxyresorufin O-deethylase (EROD), 1-chloro-2,4-dinitro-benzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) in trout liver (Oncorhynchus mykiss) compared to the controls. 2. Various treatment regimens of atrazine in a semisynthetic diet changed the XME activities towards AE, NCCR, CDNB and DCNB in the cabbage moth (Mamestra brassica L.) soft tissues and midgut compared to the controls. 3. A life-long cabbage diet induced the XME activity towards CDNB in the cabbage moth soft tissues and midgut, whereas no differences in the activities towards AE, NCCR and DCNB were observed compared to controls on a semi-synthetic diet. 4. The cabbage moth GSTs bound poorly to a glutathione (GSH)-linked epoxy-activated Sepharose 6-B; however, based on the CDNB activity recovered by a GSH elution, there were no differences in the molecular weights of the partly purified subunits (27, 26 and 25 kDa) or the pIs (5.4, 4.8, and 4.1) of the molecules in the soft tissues or midguts from respectively atrazine treated and control cabbage moth.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Characterization of multiple forms of cytochrome P-450 from an insecticide resistant strain of house fly (Musca domestica)

Abstract Components of the microsomal monooxygenase system derived from abdominal preparations of an insecticide resistant house fly ( Musca domestica , Rutgers strain) were characterized. Cytochrome P -450 isozymes with molecular weights of 49,000, 53,000, 54,000, and 58,000 were partially purified using a procedure involving chromatography on columns of phenyl Sepharose, DEAE-cellulose (DE-52), carboxymethyl-Sepharose, hydroxylapatite, and an affinity column prepared from rat liver cytochrome b 5 . Activity toward a variety of pesticides, as well as other exogenous and endogenous monooxygenase substrates, was examined using intact microsomes or purified isozymes in reconstituted systems. Oxidation of benzo[ a ]pyrene in microsomal preparations was high although neither O -dealkylation of 7-ethoxyresorufin nor N -dealkylation of p -chloromethylaniline could be detected. Other substrates metabolized by abdominal microsomes from Rutgers flies included testosterone, lauric acid (primarily at in-chain positions), phorate, p -nitroanisole, and aldrin. Activity of abdominal microsomes from the susceptible CSMA strain toward benzo[ a ]pyrene and lauric acid was, surprisingly, higher than that from the resistant Rutgers strain. Synergism between NADPH and NADH was noted with microsomes from either strain. Reconstitution experiments were relatively unsuccessful but activity toward lauric acid was obtained using a system that included purified cytochrome P -450 fractions, purified house fly or rat liver NADPH-cytochrome P -450 reductase, an NADPH generating system, and a phospholipid. Unlike similar systems utilizing mammalian cytochrome P -450, phosphatidylethanolamine was more active than phosphatidylcholine.