Inhibition of juvenile hormone biosynthesis and methyl farnesoate epoxidase activity by 1,5-disubstituted imidazoles in the cockroach,Diploptera punctata

Seventeen substituted imidazoles were tested as inhibitors of juvenile hormone (JH) III synthesis by cockroach corpora allata in an in-vitro radio-chemical assay. Most of these 1,5-disubstituted imidazoles were highly potent, with IC₅₀ values of less than 100nM. The compounds differed in their ability to cause an accumulation of the precursor methyl farnesoate in the glands. Four of the imidazoles were tested by topical application to previtellogenic adult females, and all caused a significant inhibition of JH synthesis and an accumulation of intraglandular methyl farnesoate for at least three days after treatment. Methyl farnesoate epoxidase activity of homogenates of corpora allata was inhibited by the compounds TH -14 and TH -27. This P450-dependent epoxidase activity was inhibited at less than 10 nM. The results show that the 1,5-disubstituted imidazoles are powerful inhibitors of the last step of juvenile synthesis in this cockroach.     

Molecular modifications of benzylphenol and benzyl-1,3-benzodioxole types of insect chemosterilants

Rationally planned structural modifications were carried out on benzylphenols and benzyl-1,3-benzodioxoles described as fly chemosterilants and as anti-juvenile hormones. The introduction of a prop-2-ynyloxy group at various sites of the molecule resulted in compounds with a moderate inhibitory action on cytochrome P-450 mono-oxygenases, as measured by aldrin epoxidation. One compound, 5-(4-methoxybenzyl)-6-prop-2-ynyloxy-1,3-benzodioxole, revealed chemosterilant activity on Phormia regina, but its activity was less than that of the parent compounds. 2,4-Di-tert-butyl-6-[4-(3-methoxy-3-methylbutoxy)benzyl]phenol, which possessed a juvenoid structure, revealed no juvenile hormone (JH) activity but showed a high sterilant effect against Dysdercus cingulatus. In contrast to the parent substances, none of the tested compounds showed a detectable anti-JH effect in the Galleria assay. 8-Methoxy-2,3-methylenedioxydibenz[b,e]oxepine, a hitherto undescribed fused heterocyclic ring system, was devoid of activity, indicating the importance of free rotation and/or molecular flexibility. In spite of the moderate activities of these compounds, the manifold biological potential of the quinone-methide mechanism justifies further research on these lines.     

Mixed-function oxidase activity toward cyclodiene insecticides in bass and bluegill sunfish

Microsomal mixed-function oxidase (MFO) was characterized in liver of freshwater fishes, bass, Micropterus dolomeiux, and bluegill, Lepomis macrochirus, using the chlorinated cyclodiene insecticide aldrin as a substrate.The MFO activity of bluegill adults, bluegill fry, and bass fry using cyclodienes as substrates was compared with that of mouse. Aldrin epoxidase activity was found to be high in bluegill fry (73% of the activity in mouse) compared to bluegill adults (13% of the activity in mouse).Chlordene was found to be metabolized by both fish and mouse MFO to chlordene 2,3-epoxide and 1-hydroxychlordene.Competitive inhibition between various cyclodiene substrates studied in mouse and fish showed that endrin was a potent inhibitor of aldrin epoxidation in vitro.     

Inhibition of epoxidation of methyl farnesoate to juvenile hormone III by cockroach corpus allatum homogenates

Radiolabeled methyl farnesoate is epoxidized to juvenile hormone III by an NADPH-dependent reaction occurring in corpus allatum homogenates from the cockroach Blaberus giganteus L. Most of the enzymatically produced juvenile hormone has the 10R configuration described for previously isolated natural juvenile hormones. The unnatural 2Z geometrical isomer of methyl farnesoate is epoxidized by the above system faster than the natural 2E isomer. Several series of chemicals known to be inhibitors of mixed-function oxidases were surveyed as inhibitors of methyl farnesoate epoxidation. The anti-juvenile hormone precocene II caused negligible inhibition at 1 · 10^?4M, whereas the best inhibitor was o-bromophenoxymethyl-imidazole with an apparent I₅₀ of 4 · 10^?7M. None of the inhibitors tested were potent morphogenetic agents on Tenebrio molitor pupae, and they failed to cause precocious development of Oncopeltus fasciatus nymphs. The inhibition of in vitro juvenile hormone biosynthesis suggests the possibility of finding an anti-hormone which acts by blocking juvenile hormone biosynthesis.     

The interaction of insecticide synergists with nonenzymatic model oxidation systems

Insecticide synergists such as the 1,3-benzodioxoles, the 1,2,3-benzothiadiazoles and the phenyl-2-propynyl ethers inhibit the epoxidation of aldrin to dieldrin by a modified Fenton's reagent (H₂O₂, Fe²⁺, EDTA and bovine serum albumin). Inhibition appears to result from the ability of the synergists to compete with aldrin for the OH· radicals generated by the system, and as a result of this interaction the synergists are themselves chemically modified. In the case of the 1,3-benzodioxoles the reaction results in the formation of the corresponding catechols and the rate at which this occurs correlates favorably with the ability of the synergist to inhibit aldrin epoxidation in the system. Although a number of nonenzymatic systems generating radical species other than OH· are also capable of aldrin epoxidation, these are not affected by the presence of insecticide synergists and the synergists are not themselves modified by these systems. The possible relevance of these results to the mode of action of synergists is discussed.     

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.     

Cytochrome P-450 in insects. 7. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase,and monooxygenase activities in the black blow fly (Phormia regina,Meigen)

Development and phenobarbital (PB) induction of microsomal cytochrome P-450, NADPH-cytochrome c (P-450) reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of female black blow flies (Phormia regina, Meigen). Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Induction occurred in all enzymes, even at 0.005% PB, and was maximum at 0.15%. Dramatic induction of the O-demethylation of 7-methoxy-4-methylcoumarin was observed in flies dosed with the maximum concentration of the drug. This monooxygenase activity increased to nearly 1400 times the level in control flies, whereas the other O-demethylation (methoxyresorufin) and the two epoxidation reactions exhibited considerably less change. Induction of the structural enzymes of this enzyme system were 10-fold for cytochrome P-450 and 5-fold for NADPH-cytochrome c (P-450) reductase. These data suggest that PB induces several P-450's in the blow fly, particularly one bearing a high degree of specificity for 7-methoxy-4-methycoumarin.     

Cytochrome P-450 in insects: 4. Reconstitution of cytochrome P-450-dependent monooxygenase activity in the house fly

Two cytochrome P-450-containing fractions were isolated from detergent-solubilized house fly microsomes by hydrophobic chromatography on a tryptamine-Sepharose gel. These fractions (designated P-450-1 and P-450-2) were distinctive in their spectral characteristics and in their profiles following electrophoresis in the presence of sodium dodecyl sulfate. Both fractions exhibited NADPH-dependent epoxidase activity when reconstituted with purified house fly cytochrome P-450 reductase and phospholipid. The aldrin epoxidase activity of fraction P-450-1 was twice that of P-450-2 even though heptachlor epoxidase activity of the fractions was equivalent. O-Demethylase activity with 7-methoxy-4-methylcoumarin was detectable only in the P-450-2 fraction.     

Synthesis and confirmation of structure of four mammalian metabolites of dieldrin and endrin

The first syntheses of syn-12-hydroxydieldrin, syn-12-hydroxyendrin and anti-12-hydroxyendrin have been accomplished by debenzoylation and epoxidation of three isomeric adduction products of hexachlorocyclopentadiene and 1,7,7-trinorborna-2,5-dien-7-yl benzoate. A third known metabolite of endrin, 12-ketoendrin, and a putative metabolite of dieldrin, 12-ketodieldrin, were prepared from the corresponding alcohols by oxidation with chromium trioxide. Characterisation of the three debenzoylated intermediates as syn-12-hydroxyaldrin, and syn- and anti-12-hydroxyisodrin constitutes the first syntheses of these three possible metabolites of aldrin and isodrin.     

Insect metabolism of a phenyl epoxygeranyl ether juvenoid and related compounds

1-(4′-Ethylphenoxy)-3,7-dimethyl-6,7-epoxy-trans-2-octene (the ethyl-epoxide), a potent insect morphogenetic agent, is converted to 6,7-diol and other derivatives in living cockroaches and mealworms. Enzyme preparations of these organisms, and of houseflies and several other insect species, also carry out these hydration and/or oxidation reactions. In addition, housefly microsomes epoxidize the ethyl-epoxide to a diepoxide. The diepoxide and diol are then converted by microsomes to at least six cyclic diols, probably via an epoxy-diol intermediate, the major ones being the cis- and trans-tetrahydrofurandiol derivatives. The metabolites formed by these reactions have little or no morphogenetic activity in Tenebrio assays. Attempts to find potent inhibitors for housefly epoxide hydratases were unsuccessful. The corresponding ethylphenyl geranyl ether is epoxidized by housefly microsomes, forming the more morphogenetically active ethyl-epoxide, but the major reaction is oxidation on the geranyl moiety to an unidentified olefinic carboxylic acid. The chemical modifications needed for improved stability and morphogenetic activity in this juvenoid series depend on the insect species and strain and the relative activities of their enzymes involved in various inactivation pathways.     

Studies on pesticides based on coumarin. III. Synthesis and antifungal activity of substituted 4-methyl-coumarins and related compounds

A series of substituted 4-methylcoumarins was synthesised and the members tested for their toxicity towards mycelial growth of seven phytopathogenic fungi in culture. Rhizoctonia solani, Alternaria alternata and Fusarium solani exhibited maximum sensitivity to these compounds whereas Pythium aphanidermatum, Colletotrichum falcatum, Drechslera oryzae and Macrophomina phaseolina were relatively less sensitive. 6-Ethyl-3-n- propyl-7-hydroxy4-methylcoumarin ( I ) was relatively toxic towards all fungi except C. falcatum, P. aphanidermatum and M. phaseolina. The 6-n-butyl ( III ) and 6-(1, 1, 3, 3-tetramethylbutyl) ( VI ) derivatives were highly toxic to R. solani with EC₅₀, values of lμg ml^?1.     

Inhibition of juvenile hormone biosynthesis in the tobacco hornworm, Manduca sexta, by a bisthiolcarbamate juvenoid and related compounds

Biosynthesis of juvenile hormone in the tobacco hornworm, Manduca sexta, is inhibited by the bisthiolcarbamate juvenoid N-ethyl-1,2-bis(isobutylthiolcarbamoyl)ethane both in vitro and in vivo. In vitro an extremely steep dose-response curve was obtained with an ID₅₀ value of 6 × 10^?6M. However, in vivo topical treatment with the compound resulted in mild JH antagonistic symptoms, suggesting rapid metabolism of the compound. In agreement with results from metabolic studies performed on plants and in mammals, sulfoxidation of the thiocarbamate S-(4-chlorobenzyl)N,N-diethylthiocarbamate resulted in an enhanced inhibitory effect on JH biosynthesis in vitro. This suggests that the corresponding thiocarbamate sulfoxides may act as intermediates in carbomylating critical thiol sites important in the terpenoid biosynthesis pathway. Furthermore, this study shows that these prototype compounds are interesting tools for further investigation of chemical inhibition of JH biosynthesis in insects.     

Bicyclophosphorothionate antagonists exhibiting selectivity for housefly GABA receptors

2,6,7-Trioxa-1-phosphabicyclo[2.2.2]octane 1-sulfides (bicyclophosphorothionates) with various C_1–4 alkyl groups at the 3- and 4-positions were synthesized and tested for their ability to compete with [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of γ-aminobutyric acid (GABA) receptors, for specific binding to rat-brain and housefly-head membranes, and for their insecticidal activity against houseflies. Among the 3,4-substituted analogues, 20 compounds were selectively active for housefly GABA receptors versus rat GABA receptors. The 3-alkyl groups of C₃ length and the 4-alkyl groups of C₄ length were tolerated in housefly receptors, whereas such bulky substituents were deleterious in rat receptors. The 4-isobutyl-3-isopropyl analogue was the most potent in housefly receptors (IC₅₀ = 45.2 nM ), and tert-butylbicyclophosphorothionate (TBPS), with the 4-tert-butyl group and no 3-substituent, was the most potent in rat receptors (IC₅₀ = 62.2 nM ). Their receptor selectivities (rat IC₅₀/housefly IC₅₀) were 52 and 0.038, respectively. The insecticidal activity (LD₅₀) of 20 active analogues was well correlated with their potency (IC₅₀) in inhibiting [³H]EBOB binding to housefly-head membranes (r = 0.93). The results obtained in the present study indicate that the introduction of appropriate alkyl groups into the 3- and 4-positions of bicyclophosphorothionate leads to non-competitive antagonists with increased affinity and selectivity for housefly ionotropic GABA receptors versus rat GABA_A receptors. © 1999 Society of Chemical Industry     

Effect of substituted pyrazoles and related compounds on geotropism in cress seedlings

The effects on root geotropism of three series of compounds related to 2-phenyl-8H-pyrazolo[5,1-a]isoindol-8-one (IV), its 3,3a-dihydro derivative (V) and 5-(2-carboxyphenyl)-3-phenylpyrazole (VI) have been assessed using cress seedlings. Compounds (IV) and (VI), and their derivatives, are highly active, destroying geotropism at concentrations of 10^?7-10^?9M. Substitution with various functional groups and increased molecular size resulted in only minor variation in the level of activity.     

Host plant stimulation of detoxifying enzymes in a phytophagous insect

The midgut microsomal aldrin epoxidase of variegated cutworm larvae (Peridroma saucia, Hübner) fed bean or peppermint leaves was up to 10 and 45 times more active, respectively, than that of larvae fed a basic control diet. Large increases in oxidase activity and cytochrome P-450 levels also occurred in larvae fed mint plant constituents such as menthol menthone, α-pinene, and β-pinene. Mint-fed larvae were more tolerant of the insecticide, carbaryl, than bean-fed larvae.     

Inhibitors of juvenile hormone biosynthesis in corpora allata of the cockroach Periplaneta americana (L.) in vitro

The spontaneous biosynthesis of methyl (2E,6E)-(10R)-10,11-epoxy-3,7,11-trimethyldodeca-2,6-dienoate (juvenile hormone III; JH III) in excised corpora allata of Periplaneta americana was inhibited by a number of synthetic prop-2-ynyl ethers and 1,3-benzodioxole derivatives. One structurally diverse group of compounds inhibited only the final biosynthetic enzyme methyl farnesoate epoxidase (EC.1.14.14.–) at low to moderate concentrations, but at higher concentrations, also inhibited methyl farnesoate (MF) formation, causing an accumulation of MF in the concentration range 10 to 100 μM. For a second, more limited, group of compounds, there was close congruence between the inhibition of JH III biosynthesis and that of total ester (MF plus JH III) biosynthesis over their effective inhibitory concentration ranges. In contrast to the first group, there was no accumulation of MF and these compounds evidently inhibited JH III biosynthesis, at the level of either farnesoic acid esterification by O-methyl transferase (EC.2.1.1.–), or at an earlier step in the biosynthetic pathway that remains to be elucidated.     

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.     

The breakdown of [14C]monocrotophos insecticide on maize,cabbage and apple

The breakdown of ¹⁴C-labelled monocrotophos (dimethyl 3-hydroxy-N-methyl-cis-crotonamide phosphate) on the foliage of maize, cabbage and apple and on apple fruit has been studied. The breakdown products were mainly hydrophilic compounds such as ( II ) and probably dimethyl phosphate. Other breakdown products were ( III ), ( IV ) (free and conjugated), ( VI ) and ( IX ). In maize and apple ( IV ) was conjugated with sugars other than ß-D -glucose and a polar component, possibly a conjugate, was present in cabbage but this may not have been derived from ( IV ) .     

Effects of the synergist piperonyl butoxide on metabolism of pesticides in green sunfish

The effects of piperonyl butoxide on metabolism of ¹⁴C-labeled methoxychlor, aldrin, and trifluralin were investigated in green sunfish, Lepomis cyanellus. Piperonyl butoxide inhibited epoxidation of aldrin to dieldrin, O-dealkylation of methoxychlor, and N-dealkylation of trifluralin, resulting in higher levels of total radioactivity in animals exposed to the combination compared to those exposed to pesticide alone. Where piperonyl butoxide was present a greater proportion of the total radioactivity in the fish extract occurred as parent compound compared to metabolites than in fish exposed to pesticide alone. After 16 days of exposure piperonyl butoxide increased the proportion of parent compound eight times for methoxychlor, 17 times for aldrin, and 15 times for trifluralin.