Bimodal effect of methylenedioxyphenyl compounds on detoxifying enzymes in the housefly

Thirteen methylenedioxyphenyl (MDP) compounds, including commercial insecticide synergists and juvenile hormone analogs, were compared in their effect on detoxifying enzymes in the housefly (Musca domestica). Flies were fed a diet containing 1% of the compounds for 3 days. Enzymes were then assayed in vitro for their activity using aldrin and DDT as substrates. Piperonyl butoxide (PB), sesamex, propyl isome, sulfoxide, safrole, isosafrole, 6,7-epoxy-3,7-diethyl-1-[3-4(methylenedioxy) phenoxy]-2-octene (MDP-JH I) and 6,7-epoxy-3-methyl-7-ethyl-1-[3,4-(methylenedioxy) phenoxy]-2-octene (MDP-JH II) all caused a bimodal effect, inhibiting microsomal epoxidase and inducing DDT-dehydrochlorinase in the resistant Isolan-B strain. Two of these, PB and MDP-JH I, gave similar results with the susceptible strain, stw;w⁵ and two resistant strains, Fc-B and Orlando-DDT. However, o-safrole, piperonylic acid, piperonal, 3,4-methylenedioxybenzyl acetate and methyl-(3,4-methylenedioxy) benzoate had little or no effect on the enzyme systems studied. The standard susceptible strain (WHO-SRS) responded to these compounds very differently. Among those tested, piperonyl butoxide, sesamex, safrole, and isosafrole were inducers of microsomal epoxidase, a 4-fold increase occurring after treatment with sesamex. Only MDP-JH II showed a marked inhibition of the epoxidase. These treatments did not effect DDT-dehydrochlorinase activity in this strain.The enhancement of DDT-dehydrochlorinase activity by the MDP compounds is associated with an increased rate of DDT dehydrochlorination in vivo. The stimulatory effect could be blocked by treatment with actinomycin D or cycloheximide.     

Cross resistance to juvenile hormone analogues in insecticide-resistant strains of Musca domestica L

Five juvenile hormone analogues (JHAs) were tested by topical application to prepupae of a susceptible (S) and 8 insecticide-resistant (R) strains of the housefly. Activity was measured by the inability to completely emerge from the puparium. Aitosid (isopropyl 11-methoxy-3,7,1l-trimethyldodeca-2,4-dienoate) was the most active compound against the S strain (ED₅₀ 0.0033 μg/prepupa) followed by Ro 7-9767 [6,7-epoxy-3,7-diethyl-(3,4-(methylenedioxy)phenoxy)-2-cis/trans-octene], R-20458 [trans l-(4-ethylphenoxy)-6,7-epoxy-3,7-dimethyl-2-octene], sesamex, and NIA 23509 (10,11 -epoxy-N-ethyl-3,7,11-trimethyI-2,6-dodecadienamide). The R strains, designated by the name of the selecting insecticide, have been under pressure for over 10 years and are considered maximally resistant. The dimethoate-R and OMS-15-R (carbamate-resistant) strains exhibited high levels of cross resistance to all JHAs often exceeding 100x at the ED₉₅ The fenthion-R strain showed high cross resistance toward all JHAs except Altosid, toward which it manifested an intermediate level (17.5x). The DDT/lindane-R demonstrated only negligible tolerance to Aitosid but an intermediate response to all the other JHAs. The OMS-12-R strain (phosphoramidothioate-R) exhibited intermediate to high levels of cross resistance toward all JHAs, whereas the parathion-R, Chlorthion-R and a multi-resistant field-collected strain showed only low to intermediate levels of cross resistance. On the basis of known degradative mechanisms of the OMS-15-R strain, mixed function oxidases apparently play an important role in deactivating JHAs.     

Albumin and cytochrome P450 binding characteristics of juvenile hormone and its analogs

Binding data were gathered for the cecropia juvenile hormone (methyl(E, E cis)-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate) and two of its analogs {isopropyl(2E, 4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate; (E)-4-[(6,7-epoxy-3,7-dimethyl-2-nonenyl)-oxyl]-1,2-(methylenedioxy)benzene} with bovine serum albumin and rat hepatic microsomal cytochrome P₄₅₀. The proteins were found to bind the juvenile hormone and juvenile hormone analogs with affinity constants ranging from 10⁵ to 10⁶M^?1. Thermodynamic calculations suggest that the binding of all three compounds is electrostatic in nature and that the size of the ether and ester substituents can greatly influence the binding to proteins. The juvenile hormone and its analogs all formed spectrally apparent Type I complexes with oxidized cytochrome P₄₅₀; one of the juvenile hormone analogs formed a spectrally observable product adduct with reduced cytochrome P₄₅₀. The product complex may contribute many of the hormonal effects observed for this compound.     

The metabolism of R-20458 [(E)-6,7-epoxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene] in rat hepatocyte suspensions

The metabolism of R-20458 [(E)-6,7-epoxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene] by rat hepatocytes has been analyzed and compared with that of juvenile hormone I [methyl-(E,E)-cis-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate] under identical conditions. The metabolism of R-20458 is characterized by the predominance of NADPH-dependent cytochrome P-450 and epoxide hydrolase reactions; whereas, JH I is metabolized mainly by carboxylesterase, epoxide hydrolase, and glutathione S-transferases. The metabolites of R-20458 have been shown to correspond to (E)-6,7-epoxy-1-(4-hydroxyethylphenoxy)-3,7-dimethyl-2-octene; (E)-6,7-epoxy-1-(4-acetylphenoxy)-3,7-dimethyl-2-octene; (E)-6,7-dihydroxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene; and, (E)-6,7-dihydroxy-1-(4-acetylphenoxy)-3,7-dimethyl-2-octene. The production of the α-hydroxyethyl, p-acetylphenoxy, and acetylphenoxy-6,7-diol metabolites is markedly inhibited by SKF 525-A. No dramatic effects are produced by diethylmaleate and 1,2-epoxy-3,3,3-trichloropropane.     

Effects of piperonyl butoxide and DEF on the metabolism of methoprene by the imported fire ant,Solenopsis invicta Buren

Studies are presented on the effects of two synergists, piperonyl butoxide and S,S,S-tributyl phosphorotrithioate, on the metabolism of methoprene [isopropyl (2E,4E)-11-methoxy-3,7,11-trimethyldodeca-2,4-dienoate], an insect growth regulator, by the castes of the imported fire ant, Solenopsis invicta Buren. In adults, but not in larvae, pharate pupae, and pupae, piperonyl butoxide, a microsomal enzyme inhibitor, reduced methoprene metabolism by blocking O-demethylation. S,S,S-Tributyl phosphorotrithioate, an esterase and microsomal oxidase inhibitor, was most effective in reducing methoprene metabolism in larvae. In toxicity studies, against pharate pupae, the O-demethylated methoprene metabolite (alcohol-ester) was shown to be more toxic than methoprene. Synergists may be useful in bait formulations used for imported fire ant control to extend the effectiveness of methoprene.     

Microsomal oxidase activity of three blowfly species and its induction by phenobarbital and β-naphtoflavone

Induction of the microsomal oxidase system by dietary phenobarbital and β-naphthoflavone was examined in three blowflies, Phormia regina (Mg.), Lucilia illustris (Mg.), and Eucalliphora lilica (Walk.). Responses were similar in adults and larvae of all species. Phenobarbital increased cytochrome P-450 levels up to 9-fold and aldrin epoxidase up to 138-fold. Increases in cytochrome P-450 and aldrin epoxidase caused by β-naphthoflavone were minor relative to those produced by phenobarbital. In toxicity experiments with carbaryl and propoxur tolerance was associated with the amount of microsomal oxidase activity. Using piperonyl butoxide to synergize carbaryl and propoxur there was no clear indication for the use of either the synergist ratio or synergist difference as an indicator of microsomal oxidase activity.     

The epoxidation of aldrin by microsomes from the Fc strain of housefly: A genetic survey

The DDT-resistant housefly strain, Fe, known to resist DDT by biochemical oxidation, is also resistant to carbamate insecticides and has a high in vitro microsomal epoxidase activity. The purpose of this investigation was to determine whether the DDT resistance, associated with chromosome V, is also responsible for the resistance to carbamates and for the high epoxidase levels. Genetic procedures for segregating the R factors were employed using a multimarker insecticide susceptible strain designated acbco. The technique involved backcrossing the F₁ hybrid of the resistant and susceptible parents to the susceptible parent. The genotypes with a single R chromosome from the Fc parent were retained for further development as substrains and for toxicological and biochemical studies.These studies revealed that both resistance to the carbamate insecticide, propoxur, and the high in vitro microsomal epoxidation of aldrin were lost during the genetic isolation of the R factors. However, the resistance to DDT, associated with chromosome V, was present in the substrain carrying this chromosome from the Fc parent. All of the substrains were induced five- to seven-fold, by feeding phenobarbital at 1% in the diet for 3 days.Additional substrains synthesized from the substrains carrying chromosomes II and V or III and V from the Fc parent did not possess sufficient propoxur resistance or aldrin epoxidase activity to account for that present in the R parent.The interpretation of these rseults is that neither the carbamate resistance nor the microsomal epoxidase of the Fc strain is due to the factor which oxidizes DDT. Furthermore, the factor responsible for the high microsomal epoxidase activity is not due to a single chromosome such as chromosome II which is the case in other housefly strains with high oxidase activities.     

Interactions of allelochemicals with detoxication enzymes of insecticide-susceptible and resistant fall armyworms

The induction of glutathione S-transferases and microsomal oxidases by host plants and allelochemicals was examined in sixth-instar larvae of insecticide-susceptible and resistant strains of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Among 11 host plants studied, parsnip and parsley were the best inducers of glutathione S-transferase, resulting in increases of 39- and 19-fold, respectively, compared with the artificial diet. The inducer in parsnip leaves was identified by mass spectrometry, high-pressure liquid chromatography, gas chromatography, and thin-layer chromatography as xanthotoxin, a furanocoumarin. Xanthotoxin also showed a bimodal effect on the microsomal oxidase systems, increasing cytochrome P-450 content and heptachlor epoxidase activity but inhibiting aldrin epoxidase, biphenyl 4-hydroxylase, and p-chloro-N-methylaniline N-demethylase. Using indole 3-acetonitrile, indole 3-carbinol, and flavone as inducers, the inducing pattern of glutathione S-transferases was the same toward 3,4-dichloronitrobenzene, 1-chloro-2,4-dinitrobenzene, and methyl iodide. Microsomal oxidase and glutathione S-transferase were also inducible by host plants and allelochemicals in larvae of a carbaryl-resistant strain.     

Cumene hydroperoxide-generated spectral interactions of piperonyl butoxide and other synergists with microsomes from mammals and insects

Piperonyl butoxide-dependent formation of type III difference spectra and the resulting inhibition of carbon monoxide binding by microsomal cytochrome P-450 were investigated using a cumene hydroperoxide-supplemented reaction medium. Cumene hydroperoxide is capable of supporting the formation of type III spectra with piperonyl butoxide and microsomes from several different species. NADPH is not required in the presence of cumene hydroperoxide. Similarities and differences between NADPH- and cumene hydroperoxide-mediated reactions were noted. Comparative studies indicated that, as in mammals, insect microsomal cytochrome P-450 also possesses peroxidase activity. In addition to piperonyl butoxide, other methylenedioxyphenyl compounds such as sulfoxide, n-propyl isome, and sesamol also give rise to a similar spectral response in either NADPH- or cumene hydroperoxide-supplemented reaction media. The significance of the cumene hydroperoxide-dependent reaction in elucidating the mechanism of synergistic action of methylenedioxyphenyl compounds is discussed.     

Juvenile hormone analogs: Toxicity and cross-resistance in the housefly

The toxicity of several juvenile hormone analogs (JHAs) to susceptible and insecticide-resistant housefly (Musca domestica L.) strains was determined by an assay procedure in which larvae were exposed to residues of JHAs in glass vials. All JHAs tested were toxic and the most active compound, isopropyl 11-methoxy-3, 7, 11-trimethylododeca-2, 4-dienoate, was 100 times as toxic to the susceptible Orlando Regular strain as methyl parathion and 600 times as toxic as DDT.A 5- to 30-fold tolerance to the different JHAs was present in an insecticide resistant strain in which resistance is associated with a high level of NADPH-dependent microsomal oxidase activity controlled by a gene(s) on chromosome II. Cross-resistance was less marked in a strain with a chromosome V high oxidase gene and absent in strains with other resistance mechanisms.The data indicate that cross-resistance to JHAs in insects may occur in certain strains with high levels of oxidative detoxifying activity. Even so, the most active JHA was far more toxic to both susceptible and resistant strains than methyl parathion or DDT.     

Phenobarbital induction of detoxifying enzymes in resistant and susceptible houseflies

Houseflies, Musca domestica, L., were treated with the drugs phenobarbital and 3-methylcholanthrene to study the effects of these compounds as inducing agents of the microsomal oxidases, heptachlor epoxidase, and p-nitroanisole O-demethylase, and of DDT-dehydrochlorinase. Phenobarbital was active when applied by injection or as part of the diet but inactive when topically applied. The resulting increases in heptachlor epoxidase activity were as much as 25-fold that of the untreated controls. The net increase in enzyme activity after phenobarbital treatment was greater in an insecticide-susceptible strain, WHO-SRS strain, than in a carbamate-resistant strain. However, the phenobarbital induced increases in DDT-dehydrochlorinase were greater, about 2-fold, in the resistant strains than in the susceptible strain.The optimum dose for phenobarbital was 1% in the diet for a period of 3 days. None of the treatments with 3-MC, feeding, injection, exposure to residues, or topical, were effective in induction.     

Induction of detoxifying enzymes by allelochemicals and host plants in the fall armyworm

The effect of various plant substances and host plants on the microsomal oxidases and glutathione S-transferase was investigated in the fall armyworm (Spodoptera frugiperda (J. E. Smith)) maintained on a meridic diet. The glucosinolate, sinigrin, and the hydrolytic products of glucosinolates, β-phenylethylisothiocyanate, indole 3-acetonitrile, and indole 3-carbinol, and flavone were found to be potent inducers of the glutathione S-transferase in the armyworm. An 18-fold increase in the transferase activity was observed when larvae were fed a diet containing 0.2% indole 3-acetonitrile for 2 days. These compounds, with the exception of β-phenylethylisothiocyanate which appeared to be inhibitory, also stimulated the microsomal aldrin epoxidase significantly. In all instances, no induction of the microsomal oxidase or glutathione S-transferase was observed by the plant hormones, indole 3-acetic acid and gibberellic acid; the terpenoids, stigmasterol, sitosterol, and β-carotene; the polyphenolic gossypol; and the flavonol, quercetin; some of them were found to be inhibitory. Using corn, potato, and sweet potato as inducers of various microsomal oxidases, it was found that the inducing pattern of the N-demethylase was different from the two epoxidases and O-demethylase. Corn leaves were the most active compared with other aerial parts of corn (silks, developing corn, and husk) in inducing the microsomal oxidase. The microsomal oxidase in the younger larvae appeared to be less inducible by host plants than in the older larvae.     

Use of novel approaches for designing safer drugs in insecticide research: Soft alkylating agents

Based on the analogy between anticancer drugs and insect chemosterliants, both of them acting by virtue of their alkylating capacity, chloromethyl carboxylates (described by Bodor and co-workers as potential anticancer drugs of the soft alkylating agent type) including the chloromethyl ester analogues of the juvenoids hydroprene and methoprene were prepared and tested for chemosterilant and morphogenetic juvenile hormone activity. While the chloromethyl esters of some simple carboxylic acids were inactive in this respect, the juvenoid chloromethyl esters revealed both chemosterilant and morphogenetic activity against Dysdercus cingulatus comparable with that of the known juvenoids hydroprene and methoprene. At the same time their activity against Pieris brassicae showed a sharp drop compared to the ‘parent’ compounds. The results suggest a species-dependent rate of enzymatic hydrolysis of the chloromethyl ester group as a possible reason for their selective action. Thus, the chloromethyl ester group might function as a ‘selectivity factor’).     

Microsomal oxidases in the mole crickets,Scapteriscus acletus Rehn and Hebard and Scapteriscus vicinus Scudder

An active microsomal oxidase system was found in various tissues of the mole cricket adults, Scapteriscus acletus Rehn and Hebard and Scapteriscus vicinus Scudder. Aldrin epoxidase activity was mainly located in the midgut and Malpighian tubules. p-Nitroanisole O-demethylase and p-chloro-N-methylaniline N-demethylase activities were also detected in these two tissues. The overall oxidase activities in the two species were generally similar. Seasonal variation in epoxidase activity was evident in the two species with the spring populations having more activity than the fall populations. Spectral characterization of the cytochrome P-450 revealed that the enzyme was not of “resistant type” in both species. S. vicinus was four- to fivefold more susceptible to the insecticide propoxur than S. acletus.     

A new resistance to pyrethroids in tribolium castaneum (herbst)

The characteristics of a new high-level, field-derived resistance to pyrethroids in Tribolium castaneum (Herbst) were investigated using impregnated-paper and treated-grain assays. Piperonyl butoxide almost completely suppressed the resistance, suggesting that the major resistance mechanism was microsomal oxidation. Resistance extended to all pyrethroids tested and to carbaryl but not to organophosphorus insecticides or to methoprene. Resistance was strongest against α—CN phenoxybenzyl cyclopropanecarboxylate pyrethroids and was correlated with structural modifications of the pyrethroid molecule, results also consistent with oxidative resistance. This resistance will ultimately result in failures to control T. castaneum if pyrethroids, such as deltamethrin, cypermethrin or cyfluthrin, are used in the field, even if they are synergised with piperonyl butoxide. The resistance does not jeopardise organophosphorus materials (e.g. fenitrothion, chlorpyrifos-methyl, pirimiphos-methyl, methacrifos) or methoprene.     

Metabolic O-dealkylation of 1-(4′-ethylphenoxy)-3,7-dimethyl-7-methoxy or ethoxy-trans-2-octene,potent juvenoids

1-(4′-Ethylphenoxy)-3,7-dimethyl-7-methoxy or ethoxy-trans-2-octene (the ethyl-methoxide and ethyl-ethoxide) are more stable in some biological systems and less stable in others than 1-(4′-ethylphenoxy)-3,7-dimethyl-6,7-epoxy-trans-2-octene (the ethyl-epoxide). In housefly adults and mealworm pupae the persistence increases in the order of the ethyl-epoxide, -methoxide and -ethoxide but with adult stable flies the epoxide is of intermediate stability. The alkoxides are metabolized in living insects and microsomal oxidase systems of houseflies and mouse liver mainly by O-dealkylation, at a higher rate for the methoxide than the ethoxide, but benzylic oxidation of the ethyl group also occurs and is more important in the degradation of the ethyl-epoxide than the ethyl-alkoxides. The photostability on silica gel is slightly better for the ethyl-ethoxide than the -methoxide or -epoxide.     

Comparative inhibition of the juvenile hormone esterases from Trichoplusia ni,Tenebrio molitor, and Musca domestica

Twenty-seven compounds were screened as potential inhibitors of juvenile hormone esterases. Of these compounds O-ethyl-S-phenyl phosphoramidothiolate provided the best inhibition for the cabbage looper, Trichoplusia ni (Hubner), and the yellow mealworm, Tenebrio molitor L., while the juvenile hormone esterases of the house fly, Musca domestica L., were best inhibited by a juvenoid carbamate (1-(m-phenoxy-N-ethyl carbamate)-3,7-dimethyl-7-methoxy-2E-octene). The inhibition patterns of T. ni and T. molitor are similar, while those of M. domestica are relatively different. Further studies on the juvenile hormone and α-napthyl acetate esterases of T. ni showed that they could be differentially inhibited. Diisopropyl phosphorofluoridate and an alkyl trifluoromethyl ketone selectively inhibit the hydrolysis of α-naphthyl acetate and juvenile hormone, respectively, while O-ethyl-S-phenyl phosporamidothiolate inhibits both enzymes. The juvenile hormone esterases of T. ni also appear to be unique enzymes that are selective for juvenile-hormone-like molecules. The in vivo inhibition of T. ni juvenile hormone esterases by O-ethyl-S-phenyl phosphoramidothiolate slows the in vivo hydrolysis of juvenile hormone and results in delayed pupation and malformed larvae that resemble larval-pupal intermediates. Thus, the esterases involved in juvenile hormone metabolism appear to be important in juvenile hormone regulation.     

Environmental degradation of the insect growth regulator methoprene: V. Metabolism by houseflies and mosquitoes

The metabolism of methoprene (I, isopropyl (2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate, trademark Altosid) was investigated in larval mosquitoes and houseflies. The most abundant primary metabolite in third- and fourth-instar Aedes and fourth-instar Culex larvae was the hydroxy ester while the hydroxy acid predominated in third-instar Musca larvae. Biological isomerization of the double bond at C-2 in I (i.e., conversion of (E) to (Z)) was an effective mode of insect detoxication, but these dipterans apparently cannot isomerize the (2Z) isomer of I to methoprene. In general, piperonyl butoxide and triorthocresyl phosphate slightly increased the morphogenetic activity of I in these insects.     

An Example of Cross-resistance to Pyrethroids in DDT-resistant Aedes aegypti

Comparisons of the susceptibility of several strains of adult Aedes aegypti were made. Mosquitoes from Bangkok and Jakarta were found to be highly resistant to DDT and resistant to pyrethroids relative to a laboratory strain. A strain from Singapore, where less DDT has been used, was susceptible to DDT and pyrethroids. Two strains from the Caribbean had LC₅₀ values to DDT 3 times that of the reference strain while the LC₅₀ values against bioresmethrin synergised with piperonyl butoxide were 1 1/2 times raised. Another two strains from central Africa were 2 times tolerant of DDT and 1 1/2 times tolerant of bioresmethrin plus piperonyl butoxide. Agents which block DDT-dehydrochlorinase, esterases and oxidases each caused small increases in the mortality of the Bangkok strain due to DDT and bioresmethrin as well as augmenting toxicity to the susceptible reference strain. It is tentatively suggested that resistance in the Bangkok strain is due to a combination of the actions of these and perhaps other resistance mechanisms.