Aldrin epoxidation by plant root extracts

Aldrin epoxidase activity of the cell-free pea and bean root preparations was located in the particulate fraction. High speed centrifugation at 250 000 g for 2 hr resulted in a pellet with almost all the activity of the crude cell-free preparations. While the epoxidase was stimulated by NADPH generating system in these cell-free root preparations, that in high speed centrifugation pellets was not. Aldrin epoxidase activity of the dwarf bean root homogenates was increased by addition of p-aminobenzoic acid (10^?4 M). This increase in activity is above that already manifested by Polyclar AT. No activity was detected in the dormant or germinating Alaska peas or dwarf beans until the 5th and 6th day, respectively.     

Aldrin epoxidase activity in liver microsomes from normal or streptozotocin-diabetic rats: Comparison with activity in isolated hepatocytes from normal rats incubated with glucagon

Aldrin epoxidase activity in liver microsomes from streptozotocin-diabetic rats is only 40% of that from normal rats. Epoxidation of aldrin has also been assayed in freshly isolated hepatocytes from normal rats. Addition of 10^–7 M glucagon to the incubation medium leads to a decreased aldrin epoxidase activity. Owing to the previously reported phosphorylation of a purified cytochrome P-450 isozyme, it is postulated that the cytochrome P-450 dependent aldrin epoxidase may be regulated by a glucagon induced phosphorylation process.     

Mixed function oxidases and herbivore polyphagy: the devil's advocate position

Abstract. 1. Evidence used to support the theory that mixed function oxidases (MFOs) are important in the survival of polyphagous herbivores is briefly reviewed. This evidence includes data on patterns of variation in MFO activity among species and among developmental stages within species. It also includes data on induction of MFO activity by plant compounds and metabolism of plant cornpounds by MFOs.
2. It is argued that the evidence presently available does not offer strong support of the theory because key pieces of information are lacking. Evidence which tends to refute the theory is reviewed and discussed.
3. Experiments are proposed which could more rigorously test the theory.     

CYTOCHROME P450 MONOOXYGENASES ASSOCIATED WITH PYRETHROID RESISTANCE IN HELICOVERPA ARMIGERA (HÜBNER)

Abstract  The relationship between the cytochrome P-450 (cytP⁴⁵⁰ monooxygenase and insecticide resistance mechanisms in Helicoverpa armigera was studied. The level of cytP450 and activity of p -ni-troanisole ( p -NA) O-demethylase in resistant strain were 1. 71 times and 2. 21 times respectively higher as compared with those of the susceptible strain. Aldrin epoxidase activity showed 1. 35 fold strain difference. Therefore aldrin epoxidase might not be important for H. armigera resistance mechanism, the level of cytP450 and activity of p -NA O-demethylase might play a vital role in H. armigera resistance mechanisms. The reason of different resistance mechanisms among different H. armigera resistant strains is discussed.     

棉铃虫的细胞色素P-450单加氧酶系与菊酯类杀虫剂抗药性的关系(英文)

细胞色素Ｐ－４５０单加氧酶系在昆虫抗药性中起重要作用。本文研究了棉铃虫抗性品系与敏感品系间细胞色素Ｐ４５０含量、对－硝基茴香醚Ｏ－脱甲基酶和艾氏剂环氧化酶的差异。结果表明：抗性品系中细胞色素Ｐ－４５０含量、对－硝基茴香醚Ｏ－脱甲基酶活性分别是敏感品系的１．７１倍和２．２１倍,而艾氏剂环氧化酶活性仅为１．３５倍。因此细胞色素Ｐ－４５０含量和对－硝基茴香醚Ｏ－脱甲基酶在棉铃虫抗菊酯品系中起重要作用。进一步讨论了不同抗性品系间抗性机制不同的原因。     

Spatial and temporal variation in the parasitoid assemblage of an exophytic polyphagous caterpillar

Abstract 1. Over 3400 larvae of the polyphagous ground dwelling arctiid Grammia geneura were sampled and reared over seven generations in order to characterise its parasitoid assemblage and examine how and why this assemblage varies over time and space at a variety of scales.
2. The total parasitoid assemblage of 14 species was dominated both in diversity and frequency by relatively polyphagous tachinid flies.
3. Both the composition of the parasitoid assemblage and frequency of parasitism varied strikingly among and within sampling sites, seasons, and years.
4. Overall rates of parasitism increased consistently over the duration of caterpillar development.
5. Within sampling sites, parasitism rates were non-random with respect to habitat structure and caterpillar behaviour for the most abundant parasitoid species.
6. The large variability in parasitoid assemblage structure over space and time in this system may be a function of local host population abundance, habitat-specific parasitism, and indirect interactions between G. geneura and other Macrolepidoptera through shared oligophagous and polyphagous parasitoids.     

Retinoic acid 5,6-epoxidation by hemoproteins

Retinoic acid 5,6-epoxidase activity was found in several hemoproteins such as human oxy- and methemoglobin (HbO2 and MetHb), equine skeletal muscle oxy- and metmyoglobin (MbO2 and MetMb), bovine liver catalase, and horseradish peroxidase. Hematin also catalyzed retinoic acid 5,6-epoxidation. The results suggest that the heme moiety participates in the epoxidation. However, neither horse heart cytochrome c, nor free ferrous ion nor free ferric ion exhibited the epoxidase activity. Some hemoproteins (HbO2, MetHb, MbO2, MetMb, catalase, peroxidase, and hematin) exhibited characteristic individual pH dependences of the activity, suggesting that the epoxidase activities of the hemoproteins are influenced by the apoenzymes to some degree. This view is also supported by the finding that preincubation of an HbO2 preparation at various temperatures (37-70 degrees C) reduced its epoxidase activity with increasing temperature, whereas the activity of hematin was unaffected. Active oxygen scavengers such as mannitol, catalase, and superoxide dismutase exhibited no effect on the epoxidase activities of HbO2, MetHb, MbO2, and MetMb. A ligand of heme, CN- (100 mM), inhibited the epoxidase activities but N3- (100 mM) did not. The epoxidase activities were completely inhibited by NADPH, NADH, and/or 2-mercaptoethanol but not by NADP+ and/or NAD+. An intermediate in the epoxidation may be reduced by NADPH, NADH and/or 2-mercaptoethanol. Radical species can be considered as plausible candidates for the intermediate.     

Immunoquantification of epoxide hydrolase and cytochrome P-450 isozymes in fetal and adult human liver microsomes

Epoxide hydrolase and three cytochrome P-450 isozymes were immunochemically determined in microsomes from adult and fetal human liver and tentatively correlated with some enzyme activities. The P-450 isozymes 5, 8 and 9 present in adult liver could not be positively correlated with the total cytochrome P-450 concentration spectrophotometrically determined. In fetal liver microsomes, isozyme 8 could not be detected by either electrophoretic or immunochemical procedures. Isozyme 5 was the major isozyme present in the fetal liver and its concentration increased in close relation with the total P-450 level. As shown previously, arylhydrocarbon hydroxylase activity was related to the concentration of isozyme 8 in adult liver. In fetal preparations, the absence of isozyme 8 was associated with a very low arylhydrocarbon hydroxylase activity. Aldrin epoxidase and benzphetamine-N-demethylase activities were correlated with isozyme 5 concentration, but with different slopes for adult and fetal microsomes: adult preparations catalyzed these two reactions more efficiently. Conversely, the dehydroepiandrosterone 16 beta-hydroxylase, also associated with isozyme 5 concentration, was more active in fetal than in adult microsomes. Moreover, if acetanilide hydroxylase increased with isozyme 5 concentration in adult samples, no correlation occurred between activity and P-450 isozyme level in fetal microsomes. Hydroxylations of lauric acid in positions 11 and 12 and of dehydroepiandrosterone in position 16 alpha increased with total P-450 concentration but not with isozyme concentrations whatever the age considered. Lastly, epoxide hydrolase activity towards benzopyrene 4,5-oxide was closely associated with its immunochemically determined level. These results clearly suggest that multiple mechanisms are involved in the regulation of different drug-metabolizing enzymes in the human fetus.     

The responses of polyphagous predators to prey spatial heterogeneity: aggregation by carabid and staphylinid beetles to their cereal aphid prey

Abstract. 1. Discrete patches of aphids were artificially created in winter wheat fields in 1982 and in 1983 using field cages.
2. Aggregation by polyphagous predators at these patches following cage removal was measured using pitfall traps in patch and control areas.
3. Several species of polyphagous beetle (Carabidae and Staphylinidae) aggregated in these patches while other species did not.
4. The consequences of aggregation by polyphagous predators to a single prey species are discussed.     

EFFECT OF SESAME LEAF DIET ON DETOXIFICATION ACTIVITIES OF INSECTS WITH DIFFERENT FEEDING BEHAVIOR

Three diverse insects, a polyphagous “leaf chewer” (Atractomorpha lata), a polyphagous “sap feeder” (Myzus persicae), and a “restrictive feeder” (Plutella xylostella) responded differently when fed with eight cultivars of sesame either as whole leaf or via artificial diet. There was limited or no correlation in induction between detoxifying enzyme substrates (esterase, glutathione s‐transferase [GST], and mixed function oxidase [MFO] activities) when activity toward various substrates α‐naphthyl acetate, 1‐chloro‐2,4‐dinitrobenzene, 1,2‐dichloro‐4‐nitrobenzene, and p‐nitroanisole (pNA), were compared although they were generally elevated in the tissues from insects on sesame than a reference fed with radish seedlings. In A. lata, esterase activity for the cultivar 11Pusan and 45Laos were three‐fold higher compared to the reference, while other cultivars, 24Nanbu‐twasaki and 56S‐radiatum were—two‐ to three‐fold lower than the reference. In M. persicae, the esterase activity was as much as five‐fold higher than the reference in one test cultivar. GST activities of the sesame cultivars were generally higher (≈two‐fold) than the reference in all insects and at variable ratios among the cultivars. The MFO activity toward pNA in grasshoppers feeding on these sesame cultivars was either highly expressed or nonexistent. These results indicate that although the cultivars belong to the same species, they might have undergone changes in secondary phytochemicals in response to varying biogeographical distribution. Each insect species is suspected to target a specific plant chemical burden that it tries to overcome in each cultivar through enzyme activation.     

Tellurite Specifically Affects Squalene Epoxidase: Investigations Examining the Mechanism of Tellurium-Induced Neuropathy

Abstract: A peripheral neuropathy characterized by a transient demyelinating/remyelinating sequence results when young rats are fed a tellurium-containing diet. The neuropathy occurs secondary to a systemic block in cholesterol synthesis. Squalene accumulation suggested the lesion was at the level of squalene epoxidase, a microsomal monooxygenase that uses NADPH cytochrome P450 reductase to receive its necessary reducing equivalents from NADPH. We have now demonstrated directly specificity for squalene epoxidase; our in vitro studies show that squalene epoxidase is inhibited 50% in the presence of 5 µ M tellurite, the presumptive in vivo active metabolite. Under these conditions, the activities of other monooxygenases, aniline hydroxylase and benzo( a )pyrene hydroxylase, were inhibited less than 5%. We also present data suggesting that tellurite inhibits squalene epoxidation by interacting with highly susceptible -SH groups present on this monooxygenase. In vivo studies of specificity were based on the compensatory response to feeding of tellurium. Following tellurium intoxication, there was up-regulation of squalene epoxidase activity both in liver (11-fold) and sciatic nerve (fivefold). This induction was a specific response, as demonstrated in liver by the lack of up-regulation following exposure to the nonspecific microsomal enzyme inducer, phenobarbital. As a control, we also measured the microsomal monooxygenase activities of aniline hydroxylase and benzo( a )pyrene hydroxylase. Although they were induced following phenobarbital exposure, activities of these monooxygenases were not affected following tellurium intoxication, providing further evidence of specificity of tellurium intoxication for squalene epoxidase.     

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.     

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ²⁴-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.     

Aliphatic and chlorinated alkenes and epoxides as inducers of alkene monooxygenase and epoxidase activities in Xanthobacter strain Py2.

The inducible nature of the alkene oxidation system of Xanthobacter strain Py2 has been investigated. Cultures grown with glucose as the carbon source did not contain detectable levels of alkene monooxygenase or epoxidase, two key enzymes of alkene and epoxide metabolism. Upon addition of propylene to glucose-grown cultures, alkene monooxygenase and epoxidase activities increased and after an 11-h induction period reached levels of specific activity comparable to those in propylene-grown cells. Addition of chloramphenicol or rifampin prevented the increase in the enzyme activities. Comparison of the banding patterns of proteins present in cell extracts revealed that polypeptides with molecular masses of 43, 53, and 57 kDa accumulate in propylene-grown but not glucose-grown cells. Pulse-labeling of glucose-grown cells with [35S]methionine and [35S]cysteine revealed that the 43-, 53-, and 57-kDa proteins, as well as two additional polypeptides with molecular masses of 12 and 21 kDa, were newly synthesized upon exposure of cells to propylene or propylene oxide. The addition to glucose-grown cells of a variety of other aliphatic and chlorinated alkenes and epoxides, including ethylene, vinyl chloride (1-chloroethylene), cis- and trans-1,2-dichloroethylene, 1-chloropropylene, 1,3-dichloropropylene, 1-butylene, trans-2-butylene, isobutylene, ethylene oxide, epichlorohydrin (3-chloro-1,2-epoxypropane), 1,2-epoxybutane, cis- and trans-2,3-epoxybutane, and isobutylene oxide stimulated the synthesis of the five propylene-inducible polypeptides as well as increases in alkene monooxygenase and epoxidase activities. In contrast, acetylene, and a range of aliphatic and chlorinated alkanes, did not stimulate the synthesis of the propylene-inducible polypeptides or the increase in alkene monooxygenase and epoxidase activities.     

Regulation of squalene epoxidase in HepG2 cells

Regulation of squalene epoxidase in the cholesterol biosynthetic pathway was studied in a human hepatoma cell line, HepG2 cells. Since the squalene epoxidase activity in cell homogenates was found to be stimulated by the addition of Triton X-100, enzyme activity was determined in the presence of this detergent. Incubation of HepG2 cells for 18 h with L-654,969, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, increased squalene epoxidase activity dose-dependently. On the other hand, low density lipoprotein (LDL) and 25-hydroxy-cholesterol decreased the enzyme activity. These results demonstrate that squalene epoxidase is regulated by the concentrations of endogenous and exogenous sterols. The affinity of the enzyme for squalene was not changed by treatment with L-654,969. Cytosolic (S105) fractions, prepared from HepG2 cells treated with or without L-654,969, had no effect on microsomal squalene epoxidase activity of HepG2 cells, in contrast to the stimulating effect of S105 fractions from rat liver homogenate. Mevalonate, LDL, and oxysterol treatment abolished the effect of L-654,969. Simultaneous addition of cycloheximide and actinomycin D also prevented enzyme induction in HepG2 cells. From these results, the change in squalene epoxidase activity is thought to be caused by the change in the amount of enzyme protein. It is further suggested that squalene epoxidase activity is suppressed only by sterols, not by nonsterol derivative(s) of mevalonate, in contrast to the regulation of HMG-CoA reductase.     

Life-history strategy in an oligophagous tephritid: the tomato fruit fly, Neoceratitis cyanescens

Abstract.  1. In phytophagous insects, life-history traits mainly depend on host plant range. Substantial longevity, high fecundity and larval competition are the major traits of polyphagous Tephritidae while species with a restricted host range generally exhibit a lower longevity and fecundity as well as mechanisms to avoid larval competition. Our aim in this study was to investigate the life history of an oligophagous species, the tomato fruit fly, Neoceratitis cyanescens (Bezzi).
2. We determined life tables under laboratory conditions in order to calculate the main demographic parameters of N. cyanescens and studied the influence of larval and adult diet on life-history traits.
3. The mean longevity of N. cyanescens females was 40 days. There was a strong synchronisation of female maturity. Oviposition showed an early peak at 9–13 days after a short pre-oviposition period (6 days). The absence of proteins in the adult diet both delayed ovarian maturation and decreased female fecundity. In addition, females originating from tomato fruits produced significantly more eggs than females originating from bugweed or black nightshade, showing that even the larval host plant may strongly affect the subsequent fecundity of adult females.
4. The traits of N. cyanescens are then discussed in the light of those documented for polyphagous and monophagous tephritids. Neoceratitis cyanescens displayed attributes intermediate between those of polyphagous and monophagous tephritids. Its smaller clutch size compared with polyphagous species and its specialisation on the Solanaceae family whose fruits contain toxic compounds may help in reducing intra- and inter-specific competition, respectively.     

Involvement of NADPH-cytochrome c reductase in the rat liver squalene epoxidase system.

Microsomal squalene epoxidase has previously been solubilized with Triton X-100 and resolved into fractions, FA and FB, by DEAE-cellulose chromatography (Ono T. and Bloch K (1975) J biol. Chem. 250, 1571-1579). It has now been found that FB is identical with NADPH-cytochrome c reductase (denoted FPT, EC 1.6.2.3). Although both NADPH and NADH served as electron donors, the former was preferred for squalene epoxidase activity in the reconstituted system of FA and FB. FB is characterized by its ability to reduce cytochrome c by NADPH. In place of FB, partially purified FPT was tested for its ability to support squalene epoxidation in the presence of FA. A stepwise purification of the deoxycholate-solubilized FPT yielded an increase in specific FPT activity with a parallel increase in squalene epoxidase activity. Bromelain-solubilized FPT was less effective. Rabbit antisera preparations to the purified FPT solubilized with trypsin were shown to inhibit concomitantly FPT activity and squalene epoxidase activity. These observations support the concept that squalene epoxidation is primarily mediated via a flavoprotein, NADPH-cytochrome c reductase, and a terminal oxidase, squalene epoxidase, which is distinct from cytochrome P-450.     

Oxidase activities in macrocyclic-resistant and -susceptible Haemonchus contortus

The role of oxidative metabolism in resistance to macrocyclic lactones in Haemonchus contortus was examined by measuring activities toward 2 model cytochrome P450 substrates, aldrin and ethoxycoumarin, in a susceptible and a resistant isolate of the parasite. Microsomal preparations from larvae and adults of the 2 isolates showed no differences in levels of NADPH- or cumene hydroperoxide-supported aldrin epoxidase or ethoxycoumarin O-deethylase activities. Intact adult nematodes showed an ability to catalyze the epoxidation of significant amounts of aldrin, although the nature of the enzyme group responsible was unknown. This epoxidase activity was greater in adults of the susceptible isolate. It is apparent that oxidase activities toward the 2 substrates are not enhanced in the resistant isolate, suggesting that the observed resistance to macrocyclic lactones may not involve enhanced oxidative metabolism.     

Effect of detergents on sterol synthesis in a cell-free system of yeast

In order to obtain information about the reactivity of enzymes in sterol synthesis of yeast, the effects of some detergents were investigated. Among the detergents used, Triton X-100 was found to exert a unique action, and its effect on the incorporation of 14C-labeled acetate, mevalonate, farnesyl pyrophosphate, or S-adenosyl-L-methionine into squalene, 2,3-oxidosqualene, and sterols in a cell-free system was examined. Triton X-100 showed virtually no effect on the enzyme activities in the reactions from acetyl CoA to farnesyl pyrophosphate, but it had a marked effect on reactions from farnesyl pyrophosphate to ergosterol. Evidence was obtained suggesting that Triton X-100 apparently activated squalene synthetase (EC 2.5.1.21) but inhibited squalene epoxidase (EC 1.14.99.7) and delta 24-sterol methyltransferase (EC 2.1.1.41). The activity of epoxidase was protected from the inhibition by increasing the concentration of cell-free extracts or by the prior addition of lecithin liposomes to the reaction mixture. The inhibition of methyltransferase was partially reversed by treatment with Bio-heads SM-2, but that of epoxidase was not reversed by the treatment.     

Vitamin K epoxidase activity of rough and smooth microsomes from rat liver

The distribution of vitamin K epoxidase activity in rough and smooth microsomes has been studied and compared to the prothrombin precursor and vitamin K-dependent carboxylase activity. All three activities were high in rough microsomes as compared to the low levels found in smooth microsomes. The results are in agreement with the suggestion that there might be a linkage between the vitamin K-dependent carboxylation and epoxidation reaction in vivo.