Microsomal metabolism of juvenile hormone analogs in the house fly,Musca domestica L.

Of six juvenile hormone analogs of the alkyl 3,7,11-trimethyl-2,4-dodecadienate type, only the isopropyl ester was strongly morphogenic in the house fly, Musca domestica L. In vitro assays revealed that house fly microsomes contain B-esterases as well as oxidases which metabolize such analogs. However, these esterases did not hydrolyze the isopropyl ester, ZR-515. Enzymes prepared from larvae, pupae, and adults were all active and there was evidence that in the late larval stage the esterase activity was cyclic, showing a minimum in the early third instar and a maximum a few hours later. When microsomes from two susceptible and two resistant house fly strains were compared for metabolic activity against the juvenile hormone analogs, those from the resistant strains were 1.3 to 20 × higher in oxidase activity but there was no difference in esterase activity. The oxidative metabolism of two analogs ZR-515 and 512 was greatly enhanced when the flies were induced with phenobarbital but there was no enhancement in metabolism of three of the remaining analogs and only a slight enhancement of a fourth. It is concluded that the insecticidal action of ZR-515 is largely due to its stability in the presence of the house fly esterases.     

Cytochrome P-450 in insects: 2. Multiple forms in the flesh fly (Sarcophaga bullata,Parker), and the blow fly (Phormia regina (Meigen))

Microsomes prepared from the abdomens of the flesh fly (Sarcophaga bullata, Parker) and the blow fly (Phormia regina (Meigen)) contain approximately one-fifth and one-eighth as much cytochrome P-450, respectively, as those prepared from house fly (Musca domestica, L.) abdomens. These values correlate well with the microsomal aldrin epoxidase activity of the three species and with their respective susceptibilities to the insecticide, propoxur. When the microsomes of the flesh fly and the blow fly are solubilized by treatment with deoxycholate and resolved by ion-exchange chromatography on DEAE-cellulose and hydroxylapatite, four chromatographically distinct fractions containing cytochrome P-450 are obtained. Spectrophotometric assays of the cytochrome P-450 in these fractions indicate purifications of two-to sixfold for the flesh fly hemoprotein and two-to eightfold for that of the blow fly. SDS-Polyacrylamide gel electrophoresis of the four column fractions from the flesh fly microsomes indicates that six hemoproteins in the 40,000–60,000 molecular weight range are present. In similar experiments with blow fly fractions containing approximately the same amount of cytochrome P-450 no high molecular weight hemoproteins could be detected. This result is interpreted, with other evidence, as an indication of the greater instability of the blow fly hemoprotein. The results indicate that multiple forms of cytochrome P-450 are present in both species but there is insufficient data on which to estimate the number of such forms.     

The in-vivo metabolism of propetamphos by insecticide-resistant and susceptible strains of the housefly,Musca domestica

The metabolism of propetamphos by insecticide-resistant and susceptible houseflies, in vivo, was investigated. Two major pathways of propetamphos degradation were found. The first is the major route of detoxification for both resistant and susceptible strains at low doses and involves a hydrolysis of the P–O-vinyl bond, ultimately resulting in the formation of carbon dioxide. The second major pathway involves conjugation. As the dose increases, so does the importance of this pathway. Those strains of houseflies with greater conjugative capacity are able to tolerate greater doses of propetamphos than those strains with lesser conjugative capacity. The properties exhibited by this conjugate are consistent with those of glutathione conjugates. This is further supported by a parallel between reported values of glutathione S-transferase activity in the houseflies tested and tolerance to propetamphos.     

Kinetic properties of the inhibition of juvenile hormone esterase by two trifluoromethylketones and O-ethyl,S-phenyl phosphoramidothioate

Some inhibition kinetic properties and in vivo inhibition of the plasma juvenile hormone esterase from the cabbage looper (Trichoplusia ni Hübner) by one phosphoramidothioate and two trifluoromethylketones were examined. O-ethyl,S-phenyl phosphoramidothioate was shown to react irreversibly with the enzyme in a time-dependent manner, and the inhibition reaction can be factored into a reversible step with a dissociation constant, K_d, of 4.55 × 10^?5M followed by a phosphorylation step with a rate constant, k₂, of 1.98 min^?1. The phosphorylated enzyme did not show spontaneous recovery after 48 hr of dialysis. On the other hand, the two trifluoromethylketones were shown to act as reversible inhibitors, as their inhibited enzyme was regenerated completely after dialysis. However, 1,1,1,-trifluoro-3-thiooctylpropan-2-one, in contrast to 1,1,1-trifluorotetradecan-2-one, showed progressive time-dependent inhibition, and its reaction with the enzyme followed characteristic bimolecular second-order kinetics with a rate constant, k_i, of 3.37 × 10⁷M^?1 min^?1. The in vivo inhibition data of topically treated larvae at equimolar amounts of the tested compounds indicated rapid penetration, and the stability of the inhibition was higher for the phosphoramidothioate than for the trifluoromethylketones. The relationship of the mechanism of inhibition and the in vivo inhibition of these compounds to the understanding of the interactions between juvenile hormone and juvenile hormone esterase is discussed.     

The biochemical mechanisms of resistance to insecticides with especial reference to the housefly,Musca domestica and aphid,Myzus persicae

The mechanisms by which insects chemically modify and thereby resist insecticides, and the techniques used to study this metabolism arc described. The enzymes associated with the endoplasmic reticulum of the cell (the microsomal enzymes) play a major and ubiquitous role in this metabolism which can produce molecules either more or less toxic than the insecticide applied. “Soluble” enzymes in the cytoplasm of the cell also metabolise insecticides, and it is the balance between these types of metabolism and such factors as the rate of penetration of the insecticide and the susceptibility of the target which determines the tolerance of an insect to a particular insecticide. A very small change in one of these factors can cause a large change in the dose of insecticide required to kill an insect.     

Inhibition of insect cytochrome P-450 by some metyrapone analogues and compounds containing a cyclopropylamine moiety and their evaluation as inhibitors of juvenile hormone biosynthesis

Two metyrapone analogues, 2-(l-imidazolyl)-2-methyl-l-phenyl-l-pro-panone (A-phenyl-B-imidazolyl-metyrapone; III) and 2-methyl-l-phenyl-2-{1,2,4-triazol-l-yl)-l -propanone (A-phenyl-B-triazolyl-meiyrapone; IV) as well as two cyclopropylamine derivatives. N-cyclopropyl-4-icrt-butylbenzylamine (V) and N-cyclopropyl-4-(3,7-dimethyl-7-methoxy-octyloxy)benzamide (cyclopropylamine acylated with a JH analogue acid of known structure; VI) were synthesized and evaluated in biological assays for JH biosynthesis on cockroach, Diploptera punctata corpora allata and egg growth in adult cockroach as well as for mixed function oxidase activities, i.e. epoxidation of aldrin to dieldrin and O-demethylation of 7-methoxy-4-methylcoumarin to 7-hydroxy-4-methylcoumarin on microsomes from housefly, Musca domestica, abdomen and from cockroach midgut. Compound VI was a good in-vitro inhibitor of JH biosynthesis, but it had significantly lower activities in the assays for inhibition of microsomal cytochrome P-450. Compound IV and metyrapone had moderate activity as inhibitors of oocyte growth. Compounds III, IV and V were more potent inhibitors of housefly aldrin epoxidation than metyrapone and they inhibited the enzyme activity by almost 100% at 02mM, while in cockroach midgut microsome assay metyrapone was more potent than these three compounds.     

Resistance risk assessment of two insect development inhibitors,diflubenzuron and cyromazine,for control of the housefly Musca domestica. part i: Larvicidal tests with insecticide-resistant laboratory and danish field populations

Larvicide tests with diflubenzuron (DFB) and cyromazine (CYR) were carried out against 15 laboratory housefly strains and 89 field strains collected from 87 Danish and 2 Swedish farms, 1975–89. The strains represented a wide range of adult insecticide resistance and R-mechanisms. The larvicide tests were done by treating larval medium with serial or discriminating dosages of the larvicide, seeding it with eggs and calculating the mortality during development to adults. The WHO susceptible strain (S) was used as a reference. Dose-response tests with DFB gave resistance ratios (R/S) from 1·1 to 4·1 at LC₅₀ and 0·3 to 3·4 at LC₉₅ and, with CYR, R/S from 0·6 to 1·8 at LC₅₀ and 0·6 to 2·9 at LC₉₅. It was concluded that the relatively small variation in susceptibility between strains was not generally correlated with resistance in adult flies to organophosphorus, pyrethroid or other conventional insecticides (neurotoxins). Tests with discriminating dosages of DFB (59 farms) and CYR (63 farms) showed no indication of resistance to either product. The results of investigations by other workers on the relation between resistance to DFB or CYR and resistance to conventional insecticides are discussed.     

A juvenile hormone analog, pyriproxifen, affects some biochemical components in the hemolymph and fat bodies of Eurygaster integriceps Puton (Hemiptera: Scutelleridae)

Endocrine system has a critical role during the developmental stages of insects by synthesis of several regulatory hormones. One of these hormones is juvenile hormone that several insecticides have been driven based on its biochemical structure e.g. pyriproxifen. Due to various disadvantages of fenitrothione spraying, this study was carried out finding the possible usage of pyriproxifen to control the destructive population of Eurygaster integriceps. After bioassay treatments to acquire the appropriate concentrations, the treatment repeated to find possible changes in the biochemical compounds of hemolymph and reserved macromolecules in fat bodies. Results showed significant discrepancies in amount of biochemical components of the hemolymph and the reserved macromolecules in E. integriceps after pyriproxifen treatment. In hemolymph, activity level of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase as enzymatic components and uric acid as non-enzymatic one increased but acid phosphatase, lactate dehydrogenase, protein, trehalose and lipid showed adverse results. In fat bodies, the amount of all measured reserves including glycogen, lipid and protein decreased and showed significant differences. These kinds of changes have been supported by several studies due to using insecticides. These negative effects on overall physiology of the Sunn pest by depleting the essential compounds cause sensitivity to fungal infections and several shortages for normal development and reproduction of insect. Also, the adaptability of pyriproxifen to increase the effect of entomopathogenic fungus, Beauveria bassiana, should be considered to initiate a new pest management program to decrease the production loss made by E. integriceps in wheat fields.     

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.     

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.     

Interaction of some unsubstituted phosphoramidate analogs of methamidophos (O,S-dimethyl phosphorothioamidate) with acetylcholinesterase and neuropathy target esterase of hen brain

At 37°C and pH 7.4–8.0, five higher O-alkyl analogs of methamidophos and four O-alkyl O-2,5-dichlorophenyl phosphoramidates all were more potent progressive inhibitors of hen brain AChE and neuropathy target esterase (NTE) than was methamidophos itself. For AChE, k_a increased from 7.2 × 10² to 1.0 × 10⁵ M⁻¹ min⁻¹ between methyl and n-hexyl S-methyl esters and from 9.3 × 10³ to 8.9 × 10⁵ M⁻¹ min⁻¹ between ethyl and n-hexyl dichlorophenyl analogs. For NTE, the ranges were from 16 to 7.9 × 10⁴ for S-methyl esters, and were 9.7 × 10⁴ to 7.8 × 10⁶ M⁻¹ min⁻¹ for dichlorophenyl. S-methyl esters were more active against AChE than against NTE and all the dichlorophenyl esters were more active against NTE than against AChE. Spontaneous reactivation of 75–100% activity without aging of AChE was found after 19 hr incubation at 37°C after inhibition by all nine straight-chain alkyl analogs. After inhibition by O-isopropyl S-methyl phosphorothioamidate, some spontaneous reactivation with complete aging of all remaining inhibited AChE occurred during 19 hr. No spontaneous reactivation or aging of inhibited NTE was detected. It was concluded that the molecular structures of the inhibited enzymes obtained from equivalent compounds in the two series of inhibitors were identical and that the leaving groups were, therefore, S-methyl and O-2,5-dichlorophenyl, respectively. Although hen brain NTE inhibited by methamidophos in vitro did not age, cases of delayed neuropathy in man have been reported and, presumably, require aging as well as inhibition of NTE. Possible explanations of this apparent discrepancy include (i) the fact that methamidophos consists of two chiral forms and that the form seen to be active in vitro may be disposed of preferentially in vivo, (ii) the possibility of activation in vivo to a different inhibitor, (iii) differences between conformation and ease of aging of inhibited NTE in vitro and in vivo, and (iv) species differences.     

Metabolism of some sterol inhibitors in fungi and higher plants,with special reference to the selectivity of fungicidal action

The metabolism of triforine, chloraniformethan, the α-(pyrimidin-5-yl)benzhydryl alcohols (fenarimol, nuarimol and triarimol), the trityl-azoles (fluotrimazole and clotrimazole), and the morpholine types of sterol inhibitors is reviewed; the metabolism of the azolyl-alkane derivatives (mainly triadimefon and triadimenol) is discussed in detail. Redox and hydrolytic reactions are of primary importance. Enzymic inactivation may be one factor influencing fungicide selectivity. Metabolism is the dominant factor of selectivity if it represents the activation process, as with triadimefon. Transformations in higher plants do not differ significantly from those occurring in fungi, except that factors such as the formation of conjugates with natural compounds of plant tissues also play a role, as with triforine and triadimenol. The selectivity of fungitoxic action may be influenced by metabolism both in the host plant and the pathogen.     

Determination of aldicarb, carbofuran and some of their main metabolites in groundwater by application of micellar electrokinetic capillary chromatography with diode-array detection and solid-phase extraction

This paper describes a UV detection method for the pesticides aldicarb and carbofuran, and some of their main metabolites, aldicarb-sulfoxide, aldicarb-sulfone and 3-hydroxy-carbofuran, in ground waters. Micellar electrokinetic chromatography (MEKC) with diode-array detection was developed for their determination at 210 nm. The experimental study was performed using sodium dodecyl sulfate (SDS) at a concentration level of 140 mM, and a buffer of borax/HCl 20 mM at pH 8 which gives the best resolution with an analysis time of less than 20 min. Different instrumental parameters such as voltage (23 kV), injection time (12 s) and temperature (25 degrees C) were optimized. The detection limits were in the range 2-7.4 microg glitre(-1) by solid-phase extraction (SPE) with a subsequent evaporation step. Groundwater spiked samples were pre-concentrated off-line with graphite carbon and subsequently analyzed by MEKC with diode-array detection yielding average recoveries between 77 and 97% (n = 4) with RSD between 2-7%.