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1.
A total of 185 hexanic, dichloromethanic, ethanolic and hydroethanolic extracts from 24 species of Cerrado plants, were tested against Zabrotes subfasciatus, Acanthoscelides obtectus, and human saliva α-amylases. Twelve crude extracts presented inhibition rates greater than 80% against digestive α-amylases of the insect pest Z. subfasciatus, at a concentration of 1 mg mL−1. These extracts were also tested against A. obtectus and human saliva α-amylases to verify their affinity and specificity of action. The hydroethanolic Kielmeyera coriacea stem bark extract presented a strong inhibitory potential, with IC50 values of 110 μg mL−1 for Z. subfasciatus and 272.12 μg mL−1 for A. obtectus, in addition to a 97.09% reduction in enzyme activity of human saliva α-amylases at 125 μg mL−1. The hexanic Aspidosperma macrocarpon root wood extract totally inhibited the activity of Z. subfasciatus α-amylases, reduced the enzyme activity of A. obtectus by 14.69% at 1 mg mL−1, but did not alter the activity of human saliva α-amylases, thus characterizing greater inhibition affinity and specificity. The results suggest that the application of plant extracts against insect α-amylases represent a promising biotechnological tool for development of new insect pest control strategies, with noticeable affinity and specificity of action against different target enzymes.  相似文献   

2.
Metabolism of the plant-derived phototoxic insecticide, α-terthienyl (α-T), was examined in larvae of the mosquito Culex tarsalis Coquillett (Diptera: Culicidae). Results indicated that metabolism of [3H]α-T occurred and that the resulting hydrophilic metabolites were the principal products excreted from 1-hr post-treatment onward. Pretreatment of larvae with piperonyl butoxide increased susceptibility to α-T phototoxicity and decreased the extent of [3H]α-T elimination as compared to controls. Pretreatment with β-naphthoflavone reduced α-T toxicity without affecting its elimination. Phenobarbital pretreatment affected neither toxicity nor elimination of the phototoxin. A model for interaction of Phase I and Phase II systems in metabolism of α-T by C. tarsalis larvae is proposed.  相似文献   

3.
In toxicological studies hepatocytes offer an excellent alternative to whole-animal experiments, provided their metabolic competence has been established. We have compared Phase 1 and 2 metabolism in rat, mouse, chicken and ox liver microsomes and cytosol with freshly isolated hepatocytes. The relative amounts of total cytochrome P450 in microsomes and hepatocytes were equivalent. Rat liver had the highest P450 content while chicken liver had the lowest content (148·2(±75·7) and 20·6(±11·5) pmol mg-1 hepatocellular protein, respectively). The metabolism of testosterone was assessed to determine selective cytochrome P450 isoenzyme activities. Only two metabolite products were common to all four species, namely 6β-hydroxytestosterone (6β-OHT) and androstenedione (ASD), which co-eluted with 6-dehydrotestosterone (6DHT). 16α-OHT was present in all incubations except for ox microsomes. The rate of metabolism of testosterone was generally lower in microsomes than hepatocytes, with the exception of the ox, but the pattern and quantity of metabolite formation was similar. The quantity of total products formed was 15- to 27-fold higher in rat and mouse livers than in chicken or ox. The major product formed in freshly isolated hepatocytes from mice and chickens was ASD/6DHT which accounted for 60% and 76% of the total metabolites, respectively. ASD/6DHT formation accounted for only 33% and 17% of the total metabolites formed by rat and ox hepatocytes, respectively. 2α-OHT production occurred in rat and mouse hepatocytes (14% of the total metabolites in rat and 7% in mouse hepatocytes) but was lacking in chicken or ox cells. The stability of P450 isoforms in culture was species-dependent. Rat and mouse hepatocyte cultures lost 54% and 31% of their initial P450 content after 72 h, while there was no loss in chicken hepatocytes over the same period. There was a good correlation between the relative glutathione S-transferase (GST) activities in cytosol and freshly isolated hepatocytes. Mouse liver exhibited highest GST activity (664·2(±203·5)) compared with rat, chicken or ox (320·4(±64·0), 341·5(±13·9) and 256·3(±109·9) nmol min-1 mg-1 cytosolic protein, respectively). © 1997 SCI.  相似文献   

4.
β-Pinene uncouples oxidative phosphorylation and inhibits respiration in isolated rat liver mitochondria. The uncoupling effects are observed at lower concentrations (100 to 200 μM) than the inhibition of respiration (400 μM). At low concentrations, the effects observed could be explained by an increase of the passive permeability of the mitochondrial membrane produced by the terpene. Higher concentrations seemed to inhibit respiration through an effect on the electron transport chain. At the highest concentrations tested (600 to 1200 μM), β-pinene seemed to produce a partial resealing of the mitochondrial membrane. All effects can be explained by the interaction of β-pinene with the mitochondrial membrane. Other hydrophobic molecules tested do not show the effects of β-pinene or limonene on mitochondria.  相似文献   

5.
The low mixed-function oxidase activity of house fly microsomes has been associated with low cytochrome P-450 content and NADPH-cytochrome c reductase activity. The microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity could be decreased by the addition of catechol and increased by the addition of cyanide to the homogenates. Similar results were obtained with rat liver microsomes treated with tyrosinase and catechol. During the inactivation of rat liver microsomal enzymes by tyrosinase and catechol, crosslinking of microsomal proteins occurred. These results suggest that the instability of house fly microsomal mixed-function oxidase may be due in part to the action of contaminating tyrosinase on endogenous substrates.  相似文献   

6.
Fungicides containing the imidazole and triazole groups are known to block the 14α-demethylation reaction in ergosterol biosynthesis, which is a cytochrome P-450 enzyme system. Fungicides related to diclobutrazol [(2RS, 3RS)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol] bind to cytochrome P-450 in rat liver microsomes, whole yeast cells, yeast microsomes and to a partially purified cytochrome P-450 from yeast, with Type II spectral changes. The most fungicidally active isomer (2R, 3R) shows greater binding than the less active (2S, 3S)-enantiomer to yeast microsomes; when the cytochrome P-450 was purified, a preparation was obtained to which binding more closely matched the fungicidal activity. Binding to rat liver microsomes does not reflect the fungicidal activity of the compounds.  相似文献   

7.
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.  相似文献   

8.
Crop improvement generally focuses on yield, seed quality and nutritional characteristics, rather than resistance to biotic and abiotic stresses. A clear consequence of this approach is the absence of natural anti-feedant toxins in some improved seed materials, allowing predation of commercial crops by insect herbivores. Cowpea (Vigna unguiculata), commonly cultivated by small farmers, is particularly affected by insect-pests that reproduce and develop inside stored seeds. One alternative to conventional pesticides for pest control is the use of biotechnological tools, such as the digestive enzyme inhibitors, that could be introduced in transgenic crops to enhance resistance. In this study, it was verified that the in vivo bioassays using artificial seeds containing 0.5%, 1.0% and 1.5% (w/w) of Delonix regia rich fraction, containing α-amylase inhibitors with effectiveness toward insect α-amylases and other sources, caused remarkable reduction in development and increased mortality of Callosobruchus maculatus cowpea weevil and to cotton boll weevil Anthonomus grandis. Therefore, attempts were made to isolated those inhibitors by SP-Sepharose ion exchange chromatography followed by high performance liquid chromatography on a Vydac C18-TP analytical column. Four inhibitor peaks were obtained with molecular masses of 6.0, 20 and 24 kDa. Their N-termini showed high sequence similarities with Kunitz-like inhibitor family members. These results provide evidence that D. regia synthesizes a multiple family of Kunitz-like α-amylase inhibitors, with different molecular masses and a wide biotechnological potential to control insect-pests.  相似文献   

9.
In hydroponic experiments, seed-dressing with the herbicide safener 1,8-naphthalic anhydride (NA), significantly enhanced the tolerance of maize, (Zea mays L., cv. Monarque) to the imidazolinone herbicide, AC 263222, (2-[4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl]-5-methylnicotinic acid). Uptake, distribution and metabolism studies where [14C]AC 263222 was applied through the roots of hydroponically grown maize plants showed that NA treatment reduced the translocation of radiolabel from root to shoot tissue and accelerated the degradation of this herbicide to a hydroxylated metabolite. Reductions in the lipophilicity and, therefore, mobility of this compound following hydroxylation may account for NA-induced retention of radiolabel in the root system. Hydroxylation of AC 263222 suggested that NA may stimulate the activity of enzymes involved in oxidative herbicide metabolism, such as the cytochrome P450 mono-oxygenases. In agreement with this theory, the cytochrome P450 inhibitor, 1-aminobenzotriazole (ABT), synergized AC 263222 activity and inhibited its hyroxylation in vivo. NA seed-dressing enhanced the total cytochrome P450 and b5 content of microsomes prepared from etiolated maize shoots. Isolated microsomes catalyzed AC 263222 hydroxylation in vitro. This activity possessed the characteristics of a cytochrome P450 mono-oxygenase, being NADPH-dependent and susceptible to inhibition by ABT. Activity was stimulated four-fold following NA seed treatment. Differential NA enhancement of AC 263222 hydroxylase and the cytochrome P450-dependent cinnamic acid-4-hydroxylase (CA4H) activity, suggested that separate P450 isozymes were responsible for each activity. These results indicate that the protective effects of NA result from enhancement of AC 263222 hydroxylation and concomitant reduction in herbicide translocation. This may be attributed to the stimulation of a microsomal cytochrome P450 system. © 1998 SCI.  相似文献   

10.
Pregnancy-related changes in oxidative metabolism of several xenobiotics including pesticides were examined in the hepatic microsomes of CD1 mice. The effect of pregnancy on hepatic microsomal cytochrome P-450-catalyzed substrate oxidation was found to be dependent upon the type of reaction examined. Not all substrates undergoing the same reaction showed identical changes during pregnancy. Those enzyme activities which exhibited a decline in specific activity during pregnancy generally exhibited no change in total hepatic capacity. Enzymes posting no change in specific activity throughout gestation generally showed large increases in total hepatic activity. Phorate S-oxidation was catalyzed by both microsomal flavin-containing monooxygenase (MFMO) and cytochrome P-450. Moreover, there was no pregnancy-related change in either MFMO or total enzymatic (MFMO plus cytochrome P-450) phorate S-oxidation.  相似文献   

11.
Only about 60% of the total relative gravitational force conventionally used to sediment microsomes is needed to prepare highly active microsomes from the midgut tissues of an insect larva. A rapid preliminary centrifugation for 2 min at 39,000gmax effectively removed contaminating microorganisms, tissue debris, nuclei, and mitochondria. The supernatant was recentrifuged for 20 min to 210,000g to sediment the microsomes. There were no losses of microsomal oxidase activities or degradation of cytochrome P-450 to the inactive form (P-420) resulting from the application of the higher gravitational force. Incorporation of 1 mM EDTA in the buffer and washing the microsomes resulted in an improved yield of the cytochrome compared to that in microsomes prepared in sucrose. Yields of microsomal protein, cytochrome P-450, and NADPH-cytochrome c reductase in the rapidly isolated microsomes were as good as those in conventionally prepared microsomes. The apparent kinetic characteristics of several microsomal oxidation activities and optical difference spectra of Types 1 and 2 ligands were identical in the rapidly and conventionally prepared microsomes. The morphological appearance of the microsomes was examined by electron microscopy. Microsomal pellets prepared by either method were indistinguishable. The rapid procedure saves significant time in microsome preparation and yields microsomal oxidase activities as good or slightly better than those prepared by usual centrifuged procedures.  相似文献   

12.
Maize “black Mexican sweet” (BMS) cell suspension cultures were used to study the effects of various cytochrome P450 monooxygenase inhibitors on the uptake and metabolism of the herbicide bentazon. Maize cells rapidly absorbed bentazon and metabolized it via aryl hydroxylation and glycosylation to a glycosyl conjugate of 6-hydroxybentazon. BMS cells accumulated bentazon to levels approximately 20-fold greater than those in the external medium. When BMS cells were incubated in an external medium containing 25 μM bentazon, the formation of the glycosyl conjugate (ca. 2 nmol/min/g fresh wt) was rate-limited by aryl hydroxylation. Tetcyclacis, a plant growth retardant, phenylhydrazine, a mechanism-based cytochrome P450 inhibitor, and piperonyl butoxide, an insecticide synergist, inhibited bentazon metabolism with I50 values of approximately 0.1, 1.0, and 1.0 μM, respectively. Other mechanism-based cytochrome P450 inhibitors, 3(2,4-dichlorophenoxy)-1-propyne and aminobenzotriazole, also inhibited bentazon metabolism but were less effective. The results obtained with selected inhibitors are consistent with the hypothesis that aryl hydroxylation of bentazon is catalyzed by a cytochrome P450 monooxygenase.  相似文献   

13.
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 Km'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.  相似文献   

14.
Metabolism of the herbicide thiazopyr [methyl 2-(difluoromethyl)-5-(4, 5-dihydroO-lhiazo!vt)-4-(2-methylpropy!)-6-(trinuorornethyl)-3-pyridinecarboxy-late] was examined in young seedlings of redroot pigweed, grain sorghum, sunflower, corn and soybean. As previously observed with rat liver microsomes plants predominantly metabolized thiazopyr via oxidation reactions. Sulfur and carbon atoms in the thiazoline ring were the primary sites of plant oxygenases. De-esterification was also identified as an important pathway of transformations in plants. Although similar pathways of thiazopyr metabolism were observed among plants, our data indicated species differences in rates of thiazopyr degradation. Among species examined, pigweed (Amaranthus retroflexus L.) showed the fastest metabolism. Thiazopyr metabolism in pigweed was significantly inhibited by several cytochrome P450 monooxygenase inhibitors, among which tetcyclacis (TET) and piperonyl butoxide (PBO) were the most inhibitory. Thiazopyr metabolism in pigweed was not inhibited by organophosphates, known inhibitors of esterases. The results suggest that thiazopyr metabolism in plants is predominantly mediated via plant mono-oxygenases.  相似文献   

15.
Notable differences were found among six species of wild-caught birds in the levels of cytochrome P-450, cytochrome b5, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase. Ethyl isocyanide difference spectra showed significant variations among the species in peak height and in the ratios of the 430455-nm peaks. Substantial aldrin epoxidase activity was found in all species, and the amounts of dieldrin produced compared favorably with pigeon and rat liver microsomes. Higher content of cytochrome P-450 was not always accompanied by a similar rise in specific catalytic activity. Thus, no correlation could be established between these two parameters. Aldrin epoxidase activity with NADH as the sole electron donor was 25–49% as effective as with the NADPH-generating system. Addition of both NADH and NADPH-generating systems to the incubation mixture produced a synergistic effect with liver microsomes of two species but not with two other species. DDE and polychlorinated biphenyls residues were found in the heart tissue of all species examined, and this might indicate a possible inductive effect on the microsomal mixed-function oxidase system by environmental contaminants.  相似文献   

16.
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.  相似文献   

17.
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.  相似文献   

18.
Several pesticide synergists known to be mixed-function oxidase inhibitors were found to inhibit the in vitro metabolism of diazinon by mouse liver microsomes. Piperonyl butoxide and NIA 16824 (O-isobutyl-O-propargyl phenylphosphonate) inhibit all oxidative reactions of diazinon to the same extent. In contrast, 1-(2-isopropylphenyl)imidazole selectively inhibits oxidative dearylation and thiophosphate to phosphate conversion without significant effect on ring side chain hydroxylation. This selectivity suggests that two different mechanisms of oxidative detoxification may be operating, mechanisms which may involve either two cytochrome P-450s or two different binding sites on the same cytochrome.  相似文献   

19.
Prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-imidazole-1-carboxamide), a recently developed agricultural fungicide, is a potent inducer of microsomal enzymes. Rats fed 7 days with a prochloraz-contaminated diet (2500 ppm) showed an increase in hepatic cytochrome P-450, cytochrome b5, and microsomal protein level; aniline hydroxylase, 7-ethoxycoumarin dealkylase, 7-ethoxyresorufin dealkylase, NADPH-cytochrome c reductase, and epoxide hydrolase were significantly induced. At a lower dose (100 ppm), only an increase in cytochrome P-450 and 7-ethoxyresorufin dealkylase was noticed. As shown with aniline hydroxylase and 7-ethoxycoumarin dealkylase, prochloraz is also a potent inhibitor of drug-metabolizing enzymes. The interaction of prochloraz with hepatic microsomal fraction from rat liver was also studied. Prochloraz binds to oxidized cytochrome P-450 to produce a type II spectral change; the compound also binds to reduced cytochrome P-450. The binding of some ligands (7-ethoxycoumarin, n-octylamine, aniline, and imidazole) to oxidized cytochrome P-450 was determined after induction by prochloraz. Japanese quails (Coturnix coturnix) fed 7 days with a prochloraz-contaminated diet (2000 ppm) showed a dramatic increase in liver weight (2.5-fold) and both hepatic and duodenal cytochrome P-450 (9- and 12-fold, respectively).  相似文献   

20.
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.  相似文献   

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