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. 6. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase,and monooxygenase activities in susceptible and resistant strains of Musca domestica

Development and phenobarbital (PB) induction of microsomal cytochrome P-450, cytochrome P-450 reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of insecticide-susceptible (NAIDM) and -resistant (Rutgers) house flies. Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Untreated insects of both strains had comparable reductase levels, whereas cytochrome P-450 and associated monooxygenase activities were 1.5-fold or more higher in Rutgers. Maximum induction, as well as toxicity, occurred at a lower PB concentration in NAIDM than Rutgers. The drug caused consistently higher increases in enzymes and activities within 12 hr of starting treatment in both strains. When PB was withdrawn from treated flies (both strains) 48 hr after treatment began, specific activities (product min^?1 mg protein^?1) in all enzymes returned to control values in 24 hr while metabolic capacity (product min^?1 insect^?1) achieved control values within 48 hr. The changes in turnover numbers (pmol product min^?1 pmol P-450^?1), in conjunction with the differences in the monooxygenation of the four substrates, suggest that PB treatment induced both a quantitative and qualitative change in NAIDM monooxygenation but only a quantitative change in Rutgers monooxygenation.     

Cytochrome P-450 in insects: 1. Differences in the forms present in insecticide resistant and susceptible house flies

Soluble cytochrome P-450 prepared from the microsomal fraction of abdomen homogenates of an insecticide resistant strain (Rutgers) and a susceptible strain (NAIDM) of the house fly, Musca domestica L., was characterized by spectral and electrophoretic methods. Six chromatographically distinct fractions were obtained after chromatography on DEAE-cellulose and hydroxylapatite. Examination of the six fractions by difference spectrophotometry indicated that the wave lengths for maximum absorption of the cytochrome P-450-carbon monoxide complexes were at 450, 451, and 452 nm for the NAIDM fractions and at 449, 450, and 451 nm for the Rutgers fractions. The type II binding spectra of the cytochrome P-450 in each fraction were measured with n-octylamine. Several of these resembled spectra which, in studies of hepatic cytochrome P-450, have been shown to be due to the presence of the high spin form of this hemoprotein. Four of the fractions from the resistant strain were of this type compared to one from the susceptible strain. Electrophoresis experiments indicated that there were at least three hemoproteins in the 40,000–60,000 molecular weight range in the fractions from the resistant strain while four could be detected in those from the susceptible strain. The specific aldrin epoxidase activity of the most active Rutgers fractions was considerably higher than that of similar fractions from the NAIDM microsomes in reconstitution experiments.     

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.     

Cytochrome P450 monooxygenase-mediated neonicotinoid resistance in the house fly Musca domestica L

Neonicotinoids play an essential role in the control of house flies Musca domestica. The development of neonicotinoid resistance was found in two field populations. 766b was 130- and 140-fold resistant to imidacloprid and 17- and 28-fold resistant to thiamethoxam in males and females, respectively. 791a was 22- and 20-fold resistant to imidacloprid and 9- and 23-fold resistant to thiamethoxam in males and females, respectively. Imidacloprid selection of 791a increased imidacloprid resistance to 75- and 150-fold in males and females, respectively, whereas selection with thiamethoxam had minimum impact. Neonicotinoid resistance was in all cases suppressed by PBO. The cytochrome P450 genes CYP6A1, CYP6D1 and CYP6D3 were constitutively over-expressed in resistant strains and CYP6D1 and CYP6D3 differentially expressed between sexes. The highest level of CYP6A1 expression was observed in both gender of the imidacloprid-selected strain after neonicotinoid exposure. CYP6D1 expression was increased after neonicotinoid exposure in resistant males. CYP6D3 expression was induced in both sexes upon neonicotinoid exposure but significantly higher in females.     

Blood meal and cytochrome P-450 monooxygenases in the northern house mosquito,Culex pipiens

Conditions for the measurement of aldrin epoxidation by microsomes prepared from abdominal tissues (fat body + integument) of adult female Culex pipiens were characterized. The enzyme activity had a pH optimum of 7.2 and an apparent K_m of 3.4 μM. Aldrin epoxidation and NADPH-cytochrome c reductase had similar patterns of inhibition by a rabbit antiserum to house fly NADPH-cytochrome P-450 reductase, thus implicating cytochrome P-450 monooxygenase(s) in the epoxidation of aldrin. Low (71 pmol/mg protein) levels of cytochrome P-450 were detected in abdominal tissue microsomes. In non-blood-fed insects, aldrin epoxidation and NADPH-cytochrome c reductase activities did not change between Day 1 and Day 12 after adult emergence, except for a small peak on Day 2. In insects fed a blood meal on Day 6 after emergence both activities increased (two- to threefold) to a plateau maintained between 2 and 4 days after the blood meal. Aldrin epoxidation and NADPH-cytochrome c reductase activities decreased to normal values between 4 and 6 days after the blood meal.     

Polysubstrate monooxygenases (cytochrome P-450) in larvae of susceptible and resistant strains of house flies

The polysubstrate monooxygenases (PSMO or cytochrome P-450) of house fly larvae were studied at the mature larval or “clear gut” stage. Fat body and gut tissues were most efficient in the conversion of aldrin to dieldrin. Microsomal fractions of larval homogenates had the highest PSMO activities, with lower PSMO activities also found associated with mitochondrial fractions. Microsomes from Rutgers (resistant) larvae had higher levels of NADPH:cytochrome c reductase (2×), cytochrome P-450 (2×), aldrin (4×), and heptachlor (9×) epoxidases than microsomes from CSMA (susceptible) larvae. Cytochrome P-450 of Rutgers larvae had an absorption maximum at 449 nm, 2 nm lower than the cytochrome P-450 of CSMA larvae. n-Octylamine spectra showed that the level of high-spin cytochrome P-450 was higher in Rutgers larvae. NADPH:cytochrome c reductase, cytochrome P-450, and aldrin epoxidase were induced by phenobarbital, and Rutgers larvae were shown to be more sensitive to this inducer than CSMA larvae. Induction of larval PSMO by phenobarbital did not affect the expression or the inducibility of PSMO in adults.     

Spectral interactions of insecticide synergists with microsomal cytochrome P-450 from insecticide-resistant and susceptible house flies

The spectral interactions of 45 insecticide synergists and related compounds with oxidized and reduced cytochrome P-450 from microsomes of insecticide-resistant and -susceptible house flies were investigated. The type III interaction typical of piperonyl butoxide was the most common spectral interaction for the compounds studied. In addition to this, several other varients of the type III interaction were noted. In general these responses with house fly microsomes were similar to those reported for mammals, although some minor species and strain differences were observed. The cytochrome P-450 from susceptible house flies, although reported previously not to exhibit type I difference spectra with many xenobiotics, was found to elicit this spectral response with several methylenedioxyphenyl compounds.     

Effect of the fungicide imazalil on hepatic microsomal cytochrome P-450 and cytochrome P-450 dependent activities in the Bobwhite quail (Colinus virginianus)

Bobwhite quails were treated with imazalil for 8 weeks. The fungicide was given admixed in the food at 0, 100, 300, 500 and 1000 mg kg^?1. Even at the highest dose tested, imazalil did not affect the liver weight or the hepatic microsomal protein content. In treated quails, no significant induction of cytochrome P-450 or NADPH-cytochrome c-reductase activity was observed. Furthermore, imazalil did not induce or inhibit 7-ethoxyresorufin or 7-ethoxycoumarin O-deethylase in quail microsomes. Only a slight but significant increase by 35% and 49% in aniline hydroxylase activity was measured for the 500- and 1000-mg kg^?1 dose levels, respectively. After a drug-free period of one week, aniline hydroxylase activity returned to control values, indicating that the effect was fully reversible. It is concluded that imazalil does not induce or inhibit drug-metabolizing enzymes in the quail, even at doses which exceed by far the maximum levels currently used to dress seed under field conditions (100 mg kg^?1).     

Metabolism of chlortoluron in tolerant species: possible role of cytochrome P-450 mono-oxygenases

Pathways of chlortoluron metabolism were compared in excised leaves of four tolerant species, namely wheat (Triticum aestivum var Clement), Bromus sterilis, Galium aparine and Veronica persica. The herbicide was principally detoxified by hydroxylation of the ring methyl in wheat and by N-dealkylation in Veronica persica. Both pathways were involved in Bromus sterilis and Galium aparine. Kinetic study of the degradation showed that capacity to form non-toxic conjugates could, at least partially, explain the tolerance of these species to chlortoluron. In plants treated with 1-aminobenzotriazole, a cytochrome P-450 enzyme inactivator, N-dealkylation of chlortoluron was little or not affected, but ring methyl hydroxylation was strongly inhibited. This suggests that at least two distinct enzymatic systems could participate in this metabolism. Moreover, cytochrome P-450 enzymes could be involved in the ring methyl hydroxylating reaction.     

Induction of cytochrome P450 monooxygenase activity in the two-spotted spider mite Tetranychus urticae and its influence on acaricide toxicity

Detoxification by cytochrome P450 monooxygenases is an important mechanism involved in pesticide resistance in insects and mites. The activity of these enzymes can be induced by a variety of chemicals. The aim of this study was to evaluate the effect of six P450 inducers (phenobarbital, barbital, 3-methylcholanthrene, geraniol, isosafrole, pentamethylbenzene), known to have an inducing activity in insects and mammals, on the O-deethylation activity in the two-spotted spider mite Tetranychus urticae. Treatment with barbital, phenobarbital and geraniol resulted in a dose-dependent increase in activity. Neither 3-methylcholanthrene, isosafrole nor pentamethylbenzene were effective inducers. Time course studies showed that induction by geraniol and barbital started rapidly within a period of 1-4 h after initiation of the treatment, while maximal activity was reached within 4 and 48 h, respectively. In addition, it was shown that induction with xenobiotic compounds can alter the monooxygenase-mediated acaricide tolerance in a susceptible strain of T. urticae. Although barbital induced higher levels of P450 activity, geraniol proved to be a better compound to decrease toxicity of the tested acaricides.     

Expression of a wheat cytochrome P450 monooxygenase in yeast and its inhibition by glyphosate

Glyphosate is a non-selective herbicide which acts by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase. Wheat cytochrome P450 monooxygenase specifically catalyzes the metabolism of some sulfonylurea herbicides such as chlorsulfuron and triasulfuron. Here we report that glyphosate is an inhibitor of a wheat cytochrome (CYP71C6v1), the cDNA of which was amplified by RT-PCR and heterologously expressed in yeast. The microsomal fractions derived from this strain had a Soret peak at 502 nm in the reduced carbon monoxide difference spectrum, which is a typical spectral characteristic. The addition of glyphosate to the microsomal protein resulted in a Type II spectrum indicative of binding via the nitrogen group to haem of cytochrome P450 as a sixth ligand. A spectral dissociation constant, K(s) of 70 micromol litre(-1) was observed and an IC50 of 11 micromol litre(-1) was found for glyphosate inhibition of CYP71C6v1 P450 activity.     

Expression of a wheat cytochrome P450 monooxygenase cDNA in yeast catalyzes the metabolism of sulfonylurea herbicides

A wheat cytochrome P450 cDNA (CYP71C6v1) was cloned by RT-PCR and heterologously expressed in yeast. The microsomal fractions derived from this strain could catalyze the metabolism of some sulfonylurea herbicides such as chlorsulfuron, triasulfuron, metsulfuron-metyl, bensulfuron-metyl, and tribenuron-metyl, but not sulfonylurea herbicides such as thifensulfuron and pyrazosulfuron. Kinetic parameters K_m for chlorsulfuron and triasulfuron were 57 (±15) μM and 38 (±16) μM in vitro, respectively. Analysis of the metabolites demonstrated that the CYP71C6v1 functioned as a 5-phenyl ring hydroxylase when chlorsulfuron and triasulfuron were the substrates.     

Alterations in microsomal cytochrome P-450-catalyzed reactions as a function of chlordecone (Kepone) induction

The effects of chlordecone treatment on the hepatic microsomal monooxygenase system of male rats were investigated. Chlordecone increased the microsomal content of cytochrome P-450, NADPH-cytochrome P-450 (c) reductase and, to a lesser extent, cytochrome b₅ in a time- and dose-dependent manner. The content of NADH-cytochrome b₅ (c) reductase was reduced. The turnover of seven substrates was studied in detail and, with the exception of aniline, was found to be increased between 1.3- and 2.2-fold. The apparent K_m's for these substrates were increased 2.1- to 16.7-fold. In addition, zoxazolamine paralysis time was reduced as a result of chlordecone treatment. These kinetic changes are explained on the basis of alterations in the cytochrome P-450 pool together with residual chlordecone acting as an inhibitor of substrate turnover. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein pattern of microsomes isolated from chlordecone-treated rats more closely resembled that of microsomes isolated from untreated rats than that of microsomes isolated following phenobarbital or 3-methylcholanthrene treatment.     

Effect of some ergosterol biosynthesis inhibiting fungicides on sterols and cytochrome P-450 from the Japanese quail Coturnix coturnix

Nine fungicides that inhibit ergosterol biosynthesis (diclobutrazol, fenarimol, fenpropimorph, imazalil, nuarimol, prochloraz, propiconazole, triadimefon, triadimenol) and one plant growth regulator (ancymidol) were administered to Japanese quails (Coturnix coturnix). Most of these compounds had a moderate or no effect, but prochloraz, imazalil and, to a lesser extent, propiconazole were shown to produce a dramatic increase in liver weight and cytochrome P-450 level. These three compounds were also found to be potent in-vitro inhibitors of 7-ethoxycoumarin O-de-ethylase and aniline hydroxylase, thus resulting in a biphasic effect on drug-metabolising enzymes. With these three compounds, and some others, an accumulation of lanosterol in liver was also observed, suggesting an inhibition of sterol synthesis.     

Synergism of diazinon toxicity and inhibition of diazinon metabolism in the house fly by tridiphane: Inhibition of glutathione S-transferase activity

Diazinon toxicity to a susceptible strain of house fly (Musca domestica L.) was synergized by tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane], a herbicide synergist. Both diazinon and tridiphane were partially metabolized in the house fly by glutathione (GSH) conjugation. Synergism appeared to be due to inhibition of diazinon metabolism/detoxification. Crude glutathione S-transferase (GST) preparations from the house fly catalyzed GSH conjugation of diazinon, tridiphane, 3,4-dichloronitrobenzene (DCNB), and chloro-2,4-dinitrobenzene (CDNB). Tridiphane and the GSH conjugate of tridiphane appeared to inhibit diazinon GSH conjugation, but diazinon did not inhibit tridiphane GSH conjugation. The enzymatic rate of tridiphane GSH conjugation was 22 times the rate of diazinon GSH conjugation; therefore, attempts to assay tridiphane as an inhibitor of diazinon GSH conjugation were inconclusive because of the high concentration of tridiphane GSH conjugate produced during the assay. CDNB underwent enzymatic GSH conjugation at a rate 240 times faster than that of tridiphane and 5000 times faster than that of diazinon. GSH conjugation of CDNB was not inhibited by tridiphane, but was inhibited by the GSH conjugate of tridiphane. In vivo, the GSH conjugate of tridiphane was produced in sufficient concentration to cause the observed inhibition of diazinon metabolism and synergism of diazinon toxicity. However, the possibility that parent tridiphane caused or contributed to the inhibition of diazinon metabolism and synergism of diazinon toxicity could not be excluded. Inhibition of diazinon metabolism did not appear to be due to depletion of either GSH or GST.