Changes in the activity of enzyme systems of xenobiotic metabolism at different stages of insect development

The cytochrome P-450 content, activity of microsomal monooxygenases, nonspecific esterases and glutathione S-transferases were studied at different stages of development of the Colorado beetle, cotton bollworm, cabbage butterfly, wax moth from the laboratory and natural populations. The data obtained demonstrate significant species, sexual and age differences in the activity of enzyme systems of insecticide detoxication. The toxic efficiency of insecticides at certain developmental stages depends on the level of activity of the enzyme systems involved in their metabolism. These data are discussed with respect to the problem of insects' sensitivity to insecticides at different developmental stages.     

Enzymatic systems of insecticide detoxication in a population of Colorado beetles resistant to permethrin

The activity of enzymes providing for permetrin detoxication in the imago resistant natural population of the Colorado potato beetle Leptinotarsa decemlineata was studied with the view of elucidating the biochemical mechanisms of resistance to the insecticide. It was demonstrated that the activity of the main enzymes of insect detoxication, i.e., microsomal monooxygenases, nonspecific esterases and glutathione-S-transferases in the permetrin-resistant population of L-decemlineata is enhanced as compared with the permetrin-sensitive population. It was demonstrated that the inhibitors of microsomal monooxygenases, piperonyl butoxide, and of nonspecific esterases, butifos, significantly increase the sensitivity of the resistant population to permetrin. The experimental results suggest that the activity of microsomal monooxygenases and nonspecific esterases is the main factor which determines the resistance of the Colorado potato beetle to permetrin.     

The role of enzymes of xenobiotic metabolism in the resistance of insects to insecticides

The activity of three enzymatic systems of xenobiotic metabolism (cytochrome P-450-dependent monooxygenases, non-specific esterases and glutathione S-transferases) was studied in sensitive (S) and resistant to tetrametrin (Rtetr.), permetrin (Rperm.), mecarbenyl (Rmec.) and chlorophos (Rchlor.) strains of the housefly M. domestica L. In Rtetr. and Rmec., the activity of microsomal monooxygenases was increased 2.7- and 2.3-fold, respectively, as compared to S. The position of maxima of CO-difference spectra of cytochrome P-450 in all resistant strains (with the exception of Rchlor.) were shifted towards the short-wave region by 1-2 nm. The activity of glutathione S-transferase in Rtetr. was increased as compared to S. Analysis of the total esterase activity and electrophoresis in starch gel revealed quantitative and qualitative differences between the strains under study. In all resistant strains, except for Rmec., additional bands corresponding to the esterase activity were observed. The experimental results are discussed in terms of resistance of insects to insecticides.     

Phytohemagglutinin--a modulator of activity of cytochrome P-450-dependent enzymes of hepatocyte and immunocyte membranes

The phytohemagglutinin effect in vivo on monooxygenase activity depends on the type of the cells under study (thymocytes, splenocytes, macrophages, hepatocytes) and on the period after lectin injection. Variations of monooxygenase activities associated with different forms of cytochrome P-450 are established to have a number of peculiarities in isolated hepatocytes as compared to those in the liver microsome fraction. These differences are supposed to be due to the effect of nonmicrosomal cytochrome P-450. The results obtained are discussed with regard for phytohemagglutinin effect on systems regulating activity of cytochrome P-450-dependent monooxygenases.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.     

Protein components of a cytochrome P-450 linalool 8-methyl hydroxylase

The cytochrome P-450 heme-thiolate monooxygenases that hydroxylate monoterpene hydrocarbon groups are effective models for the cytochrome P-450 family. We have purified and characterized the three proteins from a P-450-dependent linalool 8-methyl hydroxylase in Pseudomonas putida (incognita) strain PpG777. The proteins resemble the camphor 5-exohydroxylase components in chemical and physical properties; however, they show neither immunological cross-reactivity nor catalytic activity in heterogenous recombination. These two systems provide an excellent model to probe more deeply the heme-thiolate reaction center, molecular domains of substrate specificity, redox-pair interactions, and the regulation of the reaction cycle.     

艾氏剂环氧化酶及细胞色素P-450对小菜蛾抗药性发展的影响

本文对室内长期饲养的小菜蛾(Plutella xylostella L.)敏感品系和田间采集的抗性种群体内的艾氏剂环氧化酶及细胞色素P-450进行了比较研究。结果证明,艾氏剂环氧化酶在感性和抗性小菜蛾间存在着量及质的差异。 抗性种群的艾氏剂环氧化酶的Vmax和Km值分别为感性品系的5.4倍和6.5倍。抗性种群的细胞色素P-450的含量是感性品系的1.1-1.3倍。艾氏剂环氧化酶在量上及质上的差异及细胞色素P-450含量的提高是导致小菜蛾抗药性发生与发展的重要机制之一。而且质的差异较之量的差异可能起着更为重要的作用,     

艾氏剂环氧化酶及细胞色素P－450对小菜蛾抗药性发展的影响

本文对室内长期饲养的小菜蛾（Ｐｌｕｔｅｌｌａ　ｘｙｌｏｓｔｅｌｌａ　Ｌ．）敏感品系和田间采集的抗性种群体内的艾氏剂平氧化酶及细胞色素Ｐ－４５０进行了比较研究。结果证明,艾氏剂环氧化酶在感性和抗性小菜蛾间存在着量及质的差异。抗性种群的艾氏剂环氧化酶的Ｖｍａｘ和Ｋｍ值分别为感性品系的５．４％和６．５倍。抗性种群的细胞色素Ｐ－４５０的含量是感性品系的１．１—１．３倍。艾氏剂环氧化酶在量上及质上的差异及细胞色素Ｐ－４５０含量的提高是导致小菜蛾抗药性发生与发展的重要机制之一。而且质的差异较之量的差异可能起着更为重要的作用．     

Metyrapone modulation of tyrosine aminotransferase induction by dexamethasone in cultured hepatocytes

Metyrapone, an inhibitor of cytochrome P-450-dependent monooxygenases, enhanced the induction of tyrosine aminotransferase by dexamethasone in primary cultures of hepatocytes, while it had no effect on the basal level of the enzyme activity in the absence of the hormone. The amplification of the hormonal induction of tyrosine aminotransferase activity was strictly correlated with the concentration and with the inhibitory action of the compound on cytochrome P-450. The phenomenon occurred even at the maximally effective concentrations of dexamethasone, thus showing that metyrapone is a 'Glucocorticoid Potency Amplifier'. The dexamethasone activity amplification by metyrapone could be the consequence of a modulation of the glucocorticoid biotransformations due to the cytochrome P-450 inhibitor.     

Inhibition of enzymes of insecticide detoxication in insects by an alkylating analog of a cytochrome P-450 substrate

It was shown that the alkylating analog of cytochrome P-450 substrate - 4-bromomethyl-2,2,5,5-tetramethyl-3-imidazoline-3-oxide-1-oxyl effectively inhibits in vitro the activities of monooxygenases, glutathione-S-transferases and esterases from the abdomen of the house fly Musca domestica L. The non-alkylating analog (RH) appeared to be a very weak inhibitor of these enzymes. It was demonstrated that the inhibitory action of the alkylating analog consists in its covalent binding to the enzyme protein. Topical application of the cytochrome P-450 alkylating analog on insects led to a decrease of the enzyme activity by approximately 20%.     

Competition between benzo[a]pyrene and various steroids for cytochrome P-450-dependent rat liver monooxygenases

Cytochrome P-450-dependent monooxygenases are able to oxidize a large variety of endogenous and exogenous substrates. This paper describes the in vitro interaction between benzopyrene and steroids at the level of two rat liver monooxygenases: steroid-16 alpha-hydroxylase and aryl hydrocarbon hydroxylase (AHH). The results obtained suggest the following conclusions: (1) Steroid-16 alpha-hydroxylase is partially supported by a specific cytochrome P-450 form which is not inhibited in vitro by exogenous substrates. Steroid-16 alpha-hydroxylase is completely independent from cytochrome P1-450 (or P-448), as it is insensitive, in vitro, to alpha-naphthoflavone; (2) AHH is supported by two cytochrome P-450 forms: a specific form which is inducible by methylcholanthrene and inhibited in vitro by alpha-naphthoflavone, but is insensitive to metyrapone and steroids; and another less specific form which is inhibited by metyrapone and steroids in vitro.     

Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

INSECTICIDE RESISTANCE IN THE GROUND SPIDER,Pardosa sumatrana (THORELL, 1890; ARANEAE: LYCOSIDAE)

Elevated levels of insecticides detoxifying enzymes, such as esterases, glutathione S‐transferases (GSTs), and cytochrome P‐450 monooxygenases, act in the resistance mechanisms in insects. In the present study, levels of these enzymes in the insecticide‐resistant ground spider Pardosa sumatrana (Thorell, 1890) were compared with a susceptible population (control) of the same species. Standard protocols were used for biochemical estimation of enzymes. The results showed significantly higher levels of nonspecific esterases and monooxygenases in resistant spiders compared to controls. The activity of GSTs was lower in the resistant spiders. Elevated levels of nonspecific esterases and monooxygenases suggest their role in metabolic resistance in P. sumatrana. The reduced levels of total protein contents revealed its possible consumption to meet energy demands.     

Metabolic conditions determining the composition and catalytic activity of cytochrome P-450 monooxygenases in Candida tropicalis.

In the microsomal fraction of Candida tropicalis cells, two distinct monooxygenases were detected, depending on the growth conditions. The distinction of the two monooxygenases was evident from: (i) the absorption maxima in the reduced CO difference spectra of the terminal oxidases (cytochromes P-450 and P-448); (ii) the contents of the monooxygenase components (cytochromes P-450/P-448, NADPH-cytochrome c (P-450) reductase, and cytochrome b5) and (iii) the catalytic activity of the complete system (aliphatic hydroxylation and N-demethylation activity). The occurrence of the respective monooxygenases could be related to the carbon source (n-alkanes or glucose). Oxygen limitation led to a significant increase of cytochrome P-450/P-448 content, independent of the carbon source utilized by the cells. An improved method for the isolation of microsomes enabled us to demonstrate the presence of cytochrome P-448 in glucose-grown cells.     

Cartap resistance and synergism in populations of Tuta absoluta (Lep., Gelechiidae)

Abstract: Control failures of cartap when used against the tomato leafminer Tuta absoluta (Meyrick) in Brazil and a recent report of cartap resistance in Brazilian populations of this pest species led to the investigation of the possible involvement of detoxification enzymes on this phenomenon using insecticide synergists. The insect populations were collected from seven different sites in the states of Minas Gerais, Rio de Janeiro and São Paulo. These populations were subjected to insecticide-impregnated filter paper assays. The concentration–mortality assays were carried out for cartap alone and in a mixture (1 cartap : 10 synergist) with the synergists diethyl maleate, piperonyl butoxide and triphenylphosphate which, respectively, inhibit the enzymes glutathion-S-transferases, cytochrome P450-dependent monooxygenases and esterases. Resistance to cartap was observed in all populations when compared with the standard susceptible population, with resistance ratios ranging from 2.3- to 21.9-fold. Piperonyl butoxide was the most efficient synergist with cartap synergism ratios ranging from 1.3- to 21.0-fold and nearly completely suppressing the resistance to cartap in all of the populations studied, suggesting a major involvement of cytochrome P450-dependent monooxygenases as a cartap resistance mechanism in these populations of T. absoluta . Diethyl maleate and triphenylphosphate also synergized cartap in nearly every population, and they still provided partial suppression of cartap resistance in the leafminer populations studied. Therefore, glutathion-S-transferases and esterases seem to play a secondary role in cartap resistance in Brazilian populations of T. absoluta .     

Characterization of three hydroxylases involved in the final steps of biosynthesis of the steroid hormone ecdysone in Locusta migratoria (Insecta, Orthoptera)

It is most generally accepted that the last three enzymatic reactions in the biosynthetic pathway of ecdysone are, in this order, the hydroxylations at positions C-25, C-22 and C-2. Using high specific activity tritiated ecdysone precursors (2,22,25-trideoxyecdysone, 2,22-dideoxyecdysone and 2-deoxyecdysone) we have characterized the hydroxylases involved in these reactions, in the major biosynthetic tissue of ecdysone, i.e. the prothoracic glands. We show that C-2 hydroxylase is a mitochondrial oxygenase which differs from conventional cytochrome P-450-dependent monooxygenases by its relative insensitivity to CO. In contrast, C-22 and C-25 hydroxylases appear as classical cytochrome P-450 monooxygenases; C-22 hydroxylase is a mitochondrial enzyme whereas our data point to a microsomal localization of the C-25 hydroxylase.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

The role of binding in inhibition of hepatic microsomal metabolism by parathion and malathion

Parathion, malathion and their oxygenated analogs bind to the reduced form of cytochrome P-450 from rats and mice producing spectra with a maximum at 421 nm and a minimum at 450 nm. Determinations of microsomal heme suggest that the Soret at 421 nm is not associated with conversion of cytochrome P-450 to cytochrome P-420. $\text{In vivo}$ pretreatment with C¹⁴-parathion indicates that this insecticide covalently binds to mouse hepatic microsomal protein. These findings suggest that the mechanism by which these insecticides and their analogs inhibit hepatic microsomal metabolism is identical to their mode of inhibiting esterases, that is, covalent binding to catalytic site.     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.