In vitro effects of chlordecone (Kepone) on hepatic microsomal cytochrome P-450

The interaction of chlordecone (decachlorooctahydro-1,3,4-metheno-2H-cyclobuta[cd]-pentalene-2-one, Kepone) with hepatic microsomal cytochrome P-450 was studied. Chlordecone binds to cytochrome P-450 to produce a Type I spectral change the magnitude of which is dependent upon the chemical pretreatment of the animal. In kinetic studies of chlordecone was found to be a competitive inhibitor of aminopyrine N-demethylase and p-nitroanisole O-demethylase and a noncompetitive inhibitor of aniline p-hydroxylase.     

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.     

Enzyme activities and cytochrome P-450 levels of some Japanese quail tissues with respect to their metabolism of several pesticides

In the Japanese quail, cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in postmitochondrial centrifugal fractions, in microsomes, and supernatant fractions of liver, lungs, kidneys, and testes. Liver microsomes contained the highest A-esterase activity and P-450 levels. B-esterase was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in quail lung and kidney, and lowest in the liver (per mg protein). Very little difference in glutathione S-aryl transferase activity was noted among the tissues assayed. In vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed that the production of 1-naphthyl-N-hydroxymethylcarbamate and 1-naphthol, the major metabolites, was greatest in the postmitochondrial fraction of the liver. The major carbaryl metabolite in all other quail tissue fractions was 1-naphthol. Phosphamidon metabolism in postmitochondrial preparations of quail liver was higher than in the supernatant and microsomes. Chlorotoluron metabolism occurred only in the postmitochondrial fractions of quail liver. The major products were the oxidative metabolites, N-(3-chloro-4-methylphenyl)-N′-methylurea and N-(3-chloro-4-hydroxymethylphenyl)-N′-methylurea.     

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.     

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.     

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.     

Temperature and diet modulate cytochrome P-450 activities in southern armyworm,Spodoptera eridania (Cramer), caterpillars

Larvae of the southern armyworm, Spodoptera eridania (Cramer), grew well in the 15–30°C temperature range. Pupae survived poorly at 15°C but moths emerged from 85% of the pupae at 30°C. The time for development was prolonged at 15°C and larvae grew significantly bigger than at 30°C. Cytochrome P-450 content, cytochrome P-450 reductase, p-chloro N-methylaniline N-demethylation, methoxyresorufin 0-demethylation, and aldrin epoxidation activities were higher at 15°C than at 30°C. All cytochrome P-450 activities were more inducible by dietary pentamethylbenzene at 30°C than at 15°C. High cytochrome P-450-catalyzed activities were associated with increases in microsomal protein rather than with changes in membrane lipid or phospholipid content. Phosphatidylcholine was the major midgut membrane phospholipid. There was only a tendency towards increased unsaturation of the phospholipid fatty acyl moieties and lowered membrane phase transition temperature in cold-adapted larvae. Acute oral carbaryl toxicity was generally inversely correlated with cytochrome P-450 catalyzed activities. Carbaryl toxicity was decreased about 10-fold by pentamethylbenzene induction and about 3-fold by the lower acclimatization temperature.     

Methylenedioxyphenyl complexes with microsomal cytochrome P-450: In vivo complex formation in rat liver and in midgut tissues of the Southern armyworm (Spodoptera eridania)

The capacity of several methylenedioxyphenyl insecticide synergists to generate metabolite complexes with cytochrome P-450 was studied in midgut tissues of the Southern armyworm (Spodoptera eridania). Examination of the NADH-reduced versus oxidized spectra from methylene-dioxyphenyl-induced midgut indicated that isosafrole, dihydrosafrole, and 4-ethoxy-1,2-methylenedioxybenzene generated metabolite complexes with a principal absorbance maximum at 427 nm and smaller absorbance maxima near 460 and 556 nm. Further studies with 2-n-heptylbenzimidazole showed that the complex between insect cytochrome P-450 and dihydrosafrole was unusually resistant to displacement. Initial rates of complex displacement in insect microsomes were found to be approximately an order of magnitude slower than those of the corresponding complexes in rat hepatic microsomes. Nevertheless, with the exception of the dihydrosafrole complex in insect microsomes, the “time to half-maximal displacement” parameter was found to be very similar for each complex. These findings indicate that the formation of dissociable complexes between cytochrome P-450 and the methylenedioxyphenyl metabolite occurs in both insect midgut and rat hepatic microsomes after in vivo exposure. From the present study it would appear that dihydrosafrole may constitute a useful probe to distinguish binding sites within insect and mammalian cytochrome P-450.     

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.     

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.     

In-vivo effects of 2,4-D and atrazine on cytochrome P-450 and insecticide toxicity in southern armyworm (Spodoptera eridania) larvae

After feeding 2,4-D or atrazine in a diet to southern armyworm (Spodoptera eridania Cram.) larvae for three days, the effect on total content and activities of cytochrome P450 and on insecticide toxicity were determined. Both 2,4-D and atrazine induced cytochrome P450-catalyzed aldrin epoxidation (AE) and methoxyresorufin O-demethylatin (MROD). The 2,4-D was a more potent inducer for total cytochrome P450 content, whereas atrazine disproportionately increased AE. Both compounds increased MROD significantly. The apparent kinetic characteristics of AE indicates that 2,4-D and atrazine induced similar P450 isozymes (K_m 8.78 and 7.80 μM, respectively), which may differ from the constitutive isozyme (K_m 3.14 μM). The 2,4-D-induced cytochrome P450 contributed to decreased carbaryl and permethrin toxicity, whereas the atrazine-induced cytochrome P450 caused decreased parathion and permethrin toxicity. The carbaryl toxicity correlated directly with 2,4-D-induced total P450 content and activities but not with atrazine-induced changes. The 2,4-D and atrazine also induced nonspecific esterase activity which may contribute to permethrin detoxification.     

Comparative enzyme activities and cytochrome P-450 levels of some rat tissues with respect to their metabolism of several pesticides

Cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in the postmitochondrial centrifugal fraction, microsomes, and supernatant of rat liver, lungs, kidneys, and testes. Liver microsomes contained the highest P-450 levels and A-esterase activity. B-esterase activity was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in rat lung and lowest in the liver (per mg protein). Glutathione S-aryl transferase activity was highest in the liver. The in vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed many differences. Carbaryl metabolism was greater in the liver microsomal fractions than in any other preparation. 1-Naphthol was the major metabolite in all tissue fractions. Although very little metabolism of phosphamidon occurred in the rat, metabolism in the rat liver postmitochondrial fraction was slightly higher with respect to the production of metabolites than in the supernatant and microsomes combined. Chlorotoluron was not metabolized by any of the tissue fractions of the rat. At least a low level of activity toward some compounds was observed in all tissues, but this study confirmed that the liver was the most active metabolizing tissue as well as having the highest levels of enzymatic activity usually associated with pesticide metabolism.     

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.     

Observations on the antisporulant activity of imazalil (enilconazole)

Imazalil, incorporated in a solid nutrient medium, had no effect on the sporulation of Penicillium italicum at concentrations that did not strongly inhibit mycelial growth. However, when the fungus was grown submerged in liquid culture, sporulation of P. italicum appeared to be more affected than vegetative growth; at 33 nM, imazalil completely prevented the formation of penicilli without adverse effects on growth. Imazalil was able to prevent sporulation in the vapour phase as shown by scanning electron microscopy on P. italicum and Aspergillus niger; the vapours of the compound were found to affect different stages of the sporulation process, depending on the timing of the experiment. Thus, in order to show antisporulant activity, imazalil had to reach the target site by means other than migration within the fungus.     

Aims and activities of industry's fungicide resistance action committee (FRAC)1

FRAC is a GIFAP-sponsored inter-company committee dedicated to prolonging the effectiveness of fungicides liable to encounter resistance problems and to limit crop damage during the emergence of resistance. Through FRAC and the Working Groups it co-ordinates, companies are striving to establish more effective communications to alert all people involved in the research, production, marketing, registration and use of fungicides to the problems of resistance. With an enlightened attitude, effective strategies can be conceived and adopted. Co-operative action is deemed essential if the invaluable option of chemical disease control for crops is to be preserved. Working Groups for acylalanines, benzimidazoles, dicarboximides and sterol inhibitors are well organized and functioning in various monitoring studies and cooperative actions. FRAC meets regularly to review the activities of the Working Groups and to deal with the wider aspects of fungicide resistance. FRAC initiates, stimulates and monitors Working Groups, provides guidelines and co-ordinates the efforts of the Working Groups, helps Working Groups communicate their conclusions, publicises guidelines on procedures/definitions of practical resistance research, provides technical counsel for fungicide-resistance courses and research studies, and liaises with universities, advisers, farmers, distributors, governments.     

Effect of fenpropimorph and imazalil on sterol biosynthesis in penicillium italicum

Fenpropimorph was found to be highly active against Penicillium italicum (EC₅₀ 0.01/μg ml^?1). Conidia of P. italicum, treated with low concentrations of fenpropimorph, swelled in size and showed distorted germ tubes. During the initial stages of mycelial growth, fenpropimorph had little or no effect on the dry weight increase, which became strongly inhibited within 24 h after addition of the toxicant (0.05, 0.1 and 0.2 μg ml^?1). Irregular deposition of β–1,3 and β–1, 4 polysaccharides, probably chitin, was observed after treatment with fenpropimorph or imazalil. Fenpropimorph (0.05 and 0.2 μ ml^?1) caused the accumulation of a major demethyl-sterol that was different from ergosterol. It was identified as ergosta-8, 14, 24(28)-trien-3β-ol by mass, infrared, ultraviolet and proton nuclear magnetic resonance, spectrometric procedures. At both concentrations, the accumulation was already detected after incubation for 2 h. In contrast, imazalil (0.1 μg ml^?1) caused the accumulation of several methyl- and dimethyl-sterols which were tentatively identified as eburicol (24-methylene-24, 25-dihydrolanosterol), 4, 14α-dimethylergosta-8, 24(28)-dien-3-one, 14α-methylergosta-8, 24(28)-dien-3-one and obtusifoliol (4, 14α-dimethylergosta-8, 24(28)-dien-3α-ol). The accumulation of ergosta-8, 14,24(28)-trien-3β-ol indicates inhibition of the Δ¹⁴-reductase in P. italicum in a similar manner to that found previously in Ustilago maydis.     

Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi

The fungicides miconazole, fenarimol, and etaconazole block ergosterol biosynthesis in fungi by inhibiting sterol 14α-demethylation, which is mediated by a cytochrome P-450 enzyme. The sensitivity of cytochrome P-450-dependent hydroxylation or demethylation of several substrates to these fungicides and similar compounds was compared to that of fungal growth and sterol 14α-demethylation. Demethylation of p-chloro-N-methylaniline (PCMA) by sporidia of Ustilago maydis and 11α-hydroxylation of progesterone by Aspergillus nidulans were relatively insensitive to these compounds and to metyrapone. The ability of a sterol 14α-demethylation-deficient mutant to demethylate PCMA indicates that this substrate is not demethylated by the sterol 14α-demethylation system of U. maydis. The 14α-hydroxylation of progesterone by cells of Curvularia lunata was quite sensitive to the three fungicides, and also to metyrapone and isopropylphenylimidazole. This system was less sensitive to the three fungicides than sterol 14α-demethylation, but was appreciably more sensitive than PCMA demethylation. A study of progesterone 14α-hydroxylation in cell-free preparations of C. lunata showed the reaction to be inhibited by CO, and to be competitively inhibited by low concentrations of miconazole. These data suggest that the primary action of sterol biosynthesis-inhibiting (SBI) fungicides is competitive inhibition of sterol/steroid-type cytochrome P-450 enzymes rather than interference with the function of sterol carrier proteins or enzyme-modulating phospholipids.