Microsomal Monooxygenase Research Articles

Atrazine induction of a family 4 cytochrome P450 gene in Chironomus tentans (Diptera: Chironomidae)

Cytochrome P450-dependent microsomal monooxygenase (P450) activity was measured in control and atrazine-exposed third instar midge larvae, Chironomus tentans. Significantly elevated O-demethylase activity was observed in gut homogenates taken from midges exposed to atrazine concentrations from 1 to 10 ppm for 90 h. No significant induction was observed at atrazine concentrations below 1 ppm. A region of a cytochrome P450 family 4 gene was amplified and sequenced from C. tentans larvae. Alignments of inferred amino acid sequences with other insect CYP4 gene homologues indicate a high degree of similarity. Northern blot analysis employing the CYP4 gene fragment as a probe showed an over-expression in C. tentans exposed to atrazine. The results support the previously identified inducibility of cytochrome P450-dependent activity and provide insight into the potential consequences of atrazine exposure to aquatic organisms.

S-oxidation of S-methyl-esonarimod by flavin-containing monooxygenases in human liver microsomes

1. Studies using human liver microsomes and recombinant human cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) were performed to identify the enzymes responsible for the metabolism of S-methyl-esonarimod (M2), an active metabolite of esonarimod (KE-298, a novel antirheumatic drug).2. S-oxidative activities of M2 significantly correlated with those of methyl p-tolyl sulfide, a specific substrate of FMOs, as tested using 10 different human liver microsomes (r2 = 0.539, p<0.05). Thermal treatment of microsomes reduced the S-oxidative activity in the absence of the NADPH-generating system at 45°C for 5 min. However, methimazole, a known competitive substrate of FMOs, was a weak inhibitor of the S-oxidation in liver microsomes.3. Recombinant human FMO1 and FMO5 produced M3 in greater quantities than recombinant human FMO3. The S-oxidation of M2 by recombinant human FMO5 was not appreciably inhibited in the presence of methimazole. In contrast, methimazole was effective in suppressing the catalytic activity of recombinant human FMO1 and FMO3.4. The apparent Km (Km app) for the S-oxidation of M2 in human recombinant FMO5 (2.71 μM) was similar to that obtained using human liver microsomes (2.43 μM).5. The present results suggest that the S-oxidation of S-methyl esonarimod reflects FMO5 activity in the human liver because the recombinant FMO5 data match well with the human liver microsomal experiments.

Depletion of Kupffer cell function by gadolinium chloride attenuates thioacetamide-induced hepatotoxicity: Expression of metallothionein and HSP70

Kupffer cell function plays an important role in drug-induced liver injury. Thus, gadolinium chloride (GD), by selectively inactivating Kupffer cells, can alleviate drug-induced hepatotoxicity. The effect of GD was studied in reference to metallothionein and heat shock proteins expression in an in vivo model of liver necrosis induced by thioacetamide. Rats, pre-treated or not with GD (0.1 mmol/kg), were intraperitoneally injected with thioacetamide (6.6 mmol/kg), and samples of blood and liver were obtained at 0, 12, 24, 48, 72 and 96 hr. Parameters related to liver damage, Kupffer cell function, microsomal FAD monooxygenase activity, oxidative stress, and the expression of metallothionein and HSP70 were determined. GD significantly reduced serum myeloperoxidase activity and serum concentration of TNFα and IL-6, increased by thioacetamide. The extent of necrosis, the degree of oxidative stress and lipoperoxidation and microsomal FAD monooxygenase activity were significantly diminished by GD. The effect of GD induced noticeable changes in the expression of both metallothionein and HSP70, compared to those induced by thioacetamide. We conclude that GD pre-treatment reduces thioacetamide-induced liver injury and enhances the expression of metallothionein and HSP70. This effect, parallel to reduced levels of serum cytokines and myeloperoxidase activity, demonstrates that Kupffer cells are involved in thioacetamide-induced liver injury, the degree of contribution being approximately 50%.

Cytochrome P450 monooxygenases in the cotton bollworm (Lepidoptera: Noctuidae): tissue difference and induction.

Differences in the microsomal P450 monooxygenase system and its inducibility by pentamethylbenzene (PMB) and naphathalene (NA) were investigated in midgut and fatbody tissues of the cotton bollworm, Helicoverpa armigera (Boddie), larvae. Orthogonal array design was used to establish the optimal conditions for measuring Aldrin epoxidation (AE). The optima for AE were similar for the midgut and the fatbody at a temperature of 30 degrees C, pH 7.4, and a time of 10 min. In comparison to fatbody, the midgut had higher levels of total cytochrome P450s, p-nitroanisole O-demethylation (ODM) and AE. In vivo administration of 0.2% PMB or 0.2% NA resulted in higher microsomal protein content and levels of total cytochrome P450 as well as the two examined monooxygenase activities. Total cytochrome P450 and ODM activity were induced to a greater degree in the fatbody. In the midgut, PMB was significantly more effective on ODM than NA. Differences existed in SDS-PAGE profiles between the midgut and the fatbody. The induction of the midgut with PMB and of the fatbody with NA and PMB resulted in marked intensification of the protein bands with molecular masses of 59,100, 53,400, 50,400 Da.

Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase

One challenge in the heterologous expression of microsomal cytochrome P450 monooxygenases (P450s) is fulfilling their obligatory requirement for electrons transferred from NADPH P450 reductase. We have established co-expression parameters for Papilio polyxenes CYP6B1 and house fly P450 reductase in baculovirus-infected Sf9 cells that allow for efficient expression of both components and significantly enhance metabolic turnover of this insect P450’s substrates. These expression conditions have allowed us to reexamine the turnover capacities of CYP6B1 toward linear and angular furanocoumarins present in the host plants for the specialist caterpillar P. polyxenes. Coexpression of CYP6B1 and P450 reductase at equivalent viral concentrations [MOI (multiplicity of infection) ratio of 1] results in turnover rates for the linear furanocoumarins xanthotoxin and psoralen, which are increased 32–33 fold over the turnover rates obtained with CYP6B1 expressed alone. The turnover rate for the angular furanocoumarin angelicin is also significantly increased to 4.76 nmol/min/nmol P450 compared to its barely detectable level obtained with CYP6B1 expressed alone. Substrate binding analyses indicate that all three of these compounds elicit typical type I binding spectra but with varying magnitudes and affinities that are indicative of each substrate’s effectiveness at coordinating with the heme iron. The relative proportions of high spin state generated with these substrates are consistent with CYP6B1 metabolizing these furanocoumarins in the rank order xanthotoxin>psoralen>angelicin. These differential activities for CYP6B1 suggest that it may have been an ancient participant in the coevolutionary arms race between papilionid butterflies and their apiaceous host plants. Due to its ability to handle a range of furanocoumarin structures, CYP6B1 may have contributed to P. polyxenes’ early colonization of linear furanocoumarin-containing plants and to its subsequent colonization of angular furanocoumarin-containing plants.

Does the detoxification of penicillin side-chain precursors depend on microsomal monooxygenase and glutathione S-transferase in Penicillium chrysogenum?

The glutathione (GSH) S-conjugation of 1,2-epoxy-3-(4'-nitrophenoxy)propane was catalysed predominantly by microsomal glutathione S-transferase (mGST) in Penicillium chrysogenum. The specific mGST activity unlike the cytosolic GST (cGST) activity increased substantially when the penicillin side-chain precursor phenoxyacetic acid (POA) was included in the culture medium. Therefore, a microsomal monooxygenase (causing possible release of epoxide intermediates) and mGST-dependent detoxification pathway may exist for the side-chain precursors as an alternative to microsomal activation to acyl-CoA and subsequent transfer to beta-lactam molecules. The P. chrysogenum pahA and Aspergillus nidulans phacA gene products, which are cytochrome p450 monooxygenases and are able to hydroxylate phenylacetic acid (PA) at position 2 on the aromatic ring, are unlikely to release toxic epoxide intermediates but epoxidation of PA and POA due to the action of other microsomal monooxygenases cannot be excluded. The GSH-dependent detoxification of POA was provoked by a well-controlled transient lowering of pH (down to 5.0) at the beginning of the production phase in a fed-batch fermentation system. Both the specific GST and gammaGT activities were increased but the intracellular GSH concentrations remained unaltered unless the pH of the feed was transiently lowered below 5.0. At pH 4.6, the GSH pool was depleted rapidly but no antibiotic production was observed. Although sucrose was taken up effectively by the cells, cell death and autolysis were progressing. Therefore, the industrial exploitation of the GSH-dependent detoxification of penicillin side-chain precursors to reduce intracellular GSH-levels in order to avoid the GSH inhibition of the beta-lactam biosynthetic enzymes seems to be rather unlikely. P. chrysogenum mGST and cGST were separated using GSH-Sepharose 6B affinity chromatography. The purified cGST possessed a homodimer (alpha(2)) tertiary structure with M(r) (, alpha) = 29500.

Trifluoroacetylated proteins in liver and plasma of guinea pigs treated with HCFC-123 and halothane

Trifluoroacetylated (TFA)-protein adducts were investigated by immunoblotting in liver and plasma of guinea pigs treated with the hepatotoxic anaesthetic halothane or the chlorofluorocarbon replacement 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123). Male outbred Hartley guinea pigs (320–400 g) were administered HCFC-123 (1, 5, 10 and 15 mmol/kg b.w.) or halothane (positive control, 10 mmol/kg b.w.) i.p. in corn oil. Blood and liver samples were collected 24 h after administration of HCFC-123 or halothane. Immunoreactive bands were demonstrated in liver microsomes at all HCFC-123 and halothane concentrations, and in plasma of animals treated with 10 and 15 mmol/kg b.w. HCFC-123 and 10 mmol/kg b.w. halothane, while no alteration of microsomal P450 content or monooxygenase activities markers of the P450 2A, 2E1 and 2B isoforms was observed. Instead, when HCFC-123 was administered at doses of 1 and 5 mmol/kg b.w., the 2E1-dependent p-nitrophenol hydroxylase activity was enhanced. The presence of TFA-proteins in plasma was always associated with hepatic damage. However, mild liver damage in some animals treated with 1 or 5 mmol/kg b.w. HCFC-123 was not associated with the presence of TFA-proteins in plasma. This indicates a lower threshold dose for the appearance of TFA-proteins or damage in the liver (1 mmol/kg b.w.) than for the presence of TFA-proteins in plasma (10 mmol/kg b.w.), thus suggesting that the presence of TFA-proteins in plasma may be the result of liver damage.

Two forms of cytochrome P450 cDNA from 3-methylcholanthrene-treated European eel Anguilla anguilla

: The cytochrome P450 (CYP) represents a large group of microsomal monooxygenases that catalyze drugs as well as a host of lethal environmental contaminants such as dioxins, leading to either detoxification and excretion from the animal or generation of carcinogenic intermediates. In the present study two forms of cDNA were cloned (Eu MC1 and Eu MC2) for European eel CYP1A genes by polymerase chain reaction (PCR) techniques. The cDNA of Eu MC1 was 3368 bp long coding 521 amino acid residues, and that of Eu MC2 was 2464 bp long coding 517 amino acid residues. Identities of deduced amino acid sequences between Eu MC1 and Japanese eel CYP1A1 and that between Eu MC2 and the second form of Japanese eel CYP1A were 98% and 97%, respectively, showing decisively that Eu MC1 and Eu MC2 are orthologous to Japanese eel CYP1A1 and the second form of CYP1A, respectively. A striking difference between the two eel species was that the Eu MC1 peptide was two amino acid residues longer than that of the Japanese eel CYP1A1. Existence of two loci of CYP1A in Japanese and European eels may suggest that the two forms of CYP1A exist widely among the eel species, because the divergence between the two eel species has been shown to be close to the basal divergence among eels. The identities in CYP1A may help to estimate genetic distance between European and Japanese eels.

Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver, kidney and lung of CD1 mice

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL 50) mg kg −1 b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6β-hydroxylase); CYP2E1 in liver ( p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7α (14-fold) as well as 6α (22-fold), 6β, 16β and 2β hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.

Effects of the mosquito larvicide GB-1111 on red-winged blackbird embryos

Golden Bear Oil (GB-1111; legal trade name for GB-1313) is a petroleum distillate that is used in the United States and other countries as a larvicide for mosquito suppression. As part of a multi-species evaluation of the potential effects of GB-1111 on birds, red-winged blackbird eggs were collected, artificially incubated, and treated with one of five amounts of GB-1111 varying from 0 to 10 times the expected exposure from a spray application of the maximum recommended amount ( X=47 l/ha, 5 gal/ac). The application of 10 X caused a significant reduction in hatching success. A dose-related reduction of hepatic microsomal monooxygenase activity (EROD) was detected. Among body weights, skeletal measurements, and age at death, only crownrump length was different among experimental groups. Overall, the potential hazard to embryos of a representative wetland passerine appears minimal until the application rate exceeds 3 X.

Phosphorylation of Supernatant Protein Factor Enhances Its Ability to Stimulate Microsomal Squalene Monooxygenase

Supernatant protein factor is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, a downstream enzyme in the cholesterol biosynthetic pathway. The mechanism of stimulation is poorly understood, although supernatant protein factor belongs to a family of lipid-binding proteins that includes Sec14p and alpha-tocopherol transfer protein. Because recombinant human supernatant protein factor purified from Escherichia coli exhibited a relatively weak ability to activate microsomal squalene monooxygenase, we investigated the possibility that cofactors or post-translational modifications were necessary for full activity. Addition of ATP to rat liver cytosol increased supernatant protein factor activity by more than 2-fold and could be prevented by the addition of inhibitors of protein kinases A and C. Incubation of purified recombinant supernatant protein factor with ATP and protein kinases A or C delta similarly increased activity by more than 2-fold. Addition of protein phosphatase 1 gamma, a serine/threonine phosphatase, to rat liver cytosol reduced activity by 50%, suggesting that supernatant protein factor is partially phosphorylated in vivo. To determine whether dietary cholesterol influenced the phosphorylation state, cytosols were prepared from livers of rats fed a high fat diet. Although supernatant protein factor activity was reduced by more than one-half, it could not be restored by the addition of ATP or protein kinase C delta with ATP, suggesting that dietary cholesterol reduced the expression of this protein. Supernatant protein factor thus appears to be regulated both post-translationally through phosphorylation and at the level of expression. Phosphorylation may provide a means for the rapid short term modulation of cholesterol synthesis.

The CYP P450 arachidonate monooxygenases: enzymatic relays for the control of kidney function and blood pressure.

During P450-dependent metabolism, arachidonic acid (AA) is: a) hydroxylated at its ω or ω-1 carbons to 19- and 20-hydroxyeicosatetraenoic acids (HETEs)(AA ω/ω-1 hydroxylase), or b) epoxidized at any one of its four olefins to 5,6-, 8,9-, 11,12-, or 14,15-epoxyeicosatrienoic acids (EETs)(AA epoxygenase). The demonstration that many of these P450- derived eicosanoids, were formed endogenously by several rat, rabbit, and human tissues, and that they were present in human and rat plasma and/or urine, established the P450 AA monooxygenase as a formal metabolic pathway, and stimulated an extensive functional characterization of its metabolites [1, 2, 3, 4, 5, 6, 7]. During the last 15 years, the list of biological activities attributed to the P450-eicosanoids has increased at a rapid pace, and led to an extensive in vitro characterization of their vasoactive properties, roles in cell and organ ion transport, and of their participation in hormone signaling cascades [1, 2, 3, 4, 5, 6, 7]. In male Sprague-Dawley rats, AA ω/ω-1 hydroxylation and epoxidation account for 70–80, and 20–30% of the total kidney microsomal monooxygenase activity, respectively, and 20-HETE and 11(R), 12(S)-EET (88% optical purity) are recovered as the major ω-hydroxylase and epoxygenase metabolites, respectively [[3]]. The high degree of structural homology displayed by the members of a P450 gene family [8] has complicated the identification of the P450 isoforms responsible for the metabolism of endogenous AA pools [[3],[5],[9]–[11]]. Nevertheless, extensive enzymatic, biochemical, immunological, and functional evidence indicates that CYPs of the 2 and 4 gene families are responsible for AA metabolism, and that, in rat kidney, CYPs 2C and 4A are the predominant and functionally significant AA epoxygenases and ω/ω-1 hydroxylases, respectively [[3],[9]–[19]]. For example, CYPs, 2C23, 2C24, and 2C11 are expressed in the rat kidney in decreasing order of abundance [[17]] but, of these, only CYP 2C23 metabolizes AA with a regio- and enantioselectivity that matches that of the microsomal enzymes [[16],[17]]. Furthermore, as shown in Figure 1,antibodies raised against CYP 2C23 inhibited better than 95% of the microsomal epoxygenase activity present in rat kidney microsomes, and had no effect on microsomal AA ω/ω-1 hydroxylation. The recognition of CYPs 2C and 4A as endogenous AA epoxygenases and ω/ hydroxylases, as well the early proposal of a role for these isoforms in the pathophysiology of experimental hypertension [1,[4]–[7]], open the way to the introduction of molecular approaches for the study of the functional roles of the AA monooxygenases, and the characterization of P450-isoform/gene-dependent phenotypes of renal dysfunction.

Selection of Acanthoscelides obtectus (Say) for resistance to lavender essential oil vapour

A strain of A. obtectus was selected for eight generations for resistance to lavender essential oil vapour. Selection resulted in 8.6 and 4.7 times more tolerant females and males, respectively. The synergists piperonyl butoxide, diethylmaleate and triphenyl phosphate were used to gain preliminary information on the possible mechanisms of resistance. The results from synergism tests suggested that the cytochrome P450 microsomal monooxygenases and the glutathione S-transferases are involved in the observed resistance. The hypothesis of development of resistance in eggs and larvae after adult selection was also tested, but no supporting results were found.

Metabolism of tributyltin and triphenyltin by rat, hamster and human hepatic microsomes

Tributyltin and triphenyltin are metabolized by cytochrome P-450 system enzymes, and their metabolic fate may contribute to the toxicity of the chemicals. In the current study, the in vitro metabolism of tributyltin and triphenyltin by rat, hamster and human hepatic microsomes was investigated to elucidate the metabolic competence for these compounds in humans. The metabolic reaction using microsome-NADPH system that is usually conducted was not applicable to in vitro metabolism of organotins, especially triphenyltin. We therefore examined the effects of dithiothreitol (DTT), one of the antioxidants for sulfhydryl groups, to determine the in vitro metabolism of tributyltin and triphenyltin. As a result, the treatment with 0.1 mM DTT in vitro increased the activity of the microsomal monooxygenase system for metabolism of tributyltin as well as triphenyltin; the total yield of tributyltin and triphenyltin metabolites as tin increased, respectively, by approximately 1.8 and 8.9 times for rat, 2.1 and 1.2 times for hamster, and 1.6 and 1.5 times for human. It is suggested that the organotins directly inactivate cytochrome P-450 because of the interaction with critical sulfhydryl groups of the hemoprotein. We confirmed the utility of this in vitro metabolic system using DTT in the hepatic microsomes of phenobarbital (PB)-pretreated and untreated hamsters. Thus, the in vitro metabolic system described here was applied to a comparative study of the metabolism of organotins in rats, hamsters and humans. Tributyltin was metabolized more readily than triphenyltin in all the species. In humans, the in vitro metabolic pattern resembled that of hamsters, which were susceptible to in vivo triphenyltin toxicity because of incompetent metabolism. It is possible that the hamster is a qualitatively and quantitatively suitable animal model for exploring the influence of tributyltin and triphenyltin in humans.

Inhibition of cocaine oxidative metabolism attenuates endotoxin potentiation of cocaine mediated hepatotoxicity

The oxidative metabolism of cocaine by the microsomal monooxygenase enzymes has been postulated to be essential for cocaine mediated hepatotoxicity (CMH). Endotoxin (lipopolysaccharide, LPS), a well-known cause of hepatic damage, previously has been demonstrated to dramatically increase CMH. The mechanism of this interaction has not been clearly elucidated, but cocaine oxidative metabolism appears to sensitize hepatocytes so that subsequent exposure to small amounts of LPS can further augment CMH. This study was conducted to investigate if dimethylaminoethyl-2,2-diphenylvalerate (SKF-525A) pretreatment inhibits LPS potentiation of CMH. For 5 consecutive days, male CF-1 mice were administered daily SKF-525A (50 mg/kg) or sterile saline followed an hour later by cocaine (20 mg/kg) or sterile saline. Four hours following the last cocaine or saline treatment, the mice were administered sterile saline 12×10 6 EU LPS/kg, i.p. The mice were sacrificed 18 h later by decapitation. Pretreatment with SKF-525A reversed the hepatic injury caused by cocaine alone or cocaine and LPS treatments, as indicated by both histologic evaluation and serum alanine transaminase (ALT) and aspartate transaminase (AST) activities. In particular, SKF-525A completely reversed the effects of cocaine alone on liver and blood reduced gluthathione (GSH), glutathione peroxidase (GPx) and catalase (CAT) and hepatic glutathione reductase (GRx) activities. However, SKF-525A was ineffective against the effect of LPS alone on liver and blood GPx and CAT or on hepatic GSH and GRx, suggesting that these effects were not mediated by cytochrome P450 oxidative metabolism. The pattern of biochemical changes persisting with SKF-525A pretreatment in the LPS and cocaine group resembled those of the LPS alone group. The results suggest that cytochrome P450 oxidative metabolism of cocaine is largely responsible for CMH with potentiation by LPS achieved through a different mechanism involving oxidative stress.

Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster.

Five different enzymatic activities, catalyzed by both microsomal and mitochondrial cytochrome P450 monooxygenases (CYPs), are strongly implicated in the biosynthesis of ecdysone (E) from cholesterol. However, none of these enzymes have been characterized completely. The present data show that the wild-type genes of two members of the Halloween family of embryonic lethals, disembodied (dib) and shadow (sad), code for mitochondrial cytochromes P450 that mediate the last two hydroxylation reactions in the ecdysteroidogenic pathway in Drosophila, namely the C22- and C2-hydroxylases. When sad (CYP315A1) is transfected into Drosophila S2 cells, the cells metabolize 2-deoxyecdysone (2dE) to E and the [3H]ketotriol (2,22-dideoxyecdysone) to 22-deoxyecdysone. In contrast, dib (CYP302A1) is responsible for the conversion of the [3H]ketotriol to [3H]2dE. When cells are transfected with both dib and sad, they metabolize the [3H]ketotriol to [3H]E in high yield. The expression of sad and dib is concentrated within the individual segments of the developing epidermis when there is a surge of ecdysteroid midway through embryogenesis. This result occurs before the ring gland has developed and suggests that the embryonic epidermis is a site of ecdysteroid biosynthesis. This pattern then diminishes, and, during late embryogenesis, expression of both genes is concentrated in the prothoracic gland cells of the developing ring gland. Expression of dib and sad continues to be localized in this endocrine compartment during larval development, being maximal in both the late second and third instar larvae, about the time of the premolt peaks in the ecdysteroid titer.

Toxicological and mechanistic studies on neonicotinoid cross resistance in Q-type Bemisia tabaci (Hemiptera: Aleyrodidae).

The tobacco whitefly, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) is a serious pest in numerous cropping systems and has developed a high degree of resistance against several chemical classes of insecticides. One of the latest group of insecticides introduced to the market were the neonicotinoids (chloronicotinyls), acting agonistically on insect nicotinic acetylcholine receptors. Resistance to neonicotinoid insecticides has recently been shown to occur, especially in Q-type B tabaci in some places in Almeria, Spain, whereas control of B-type B tabaci in many other intense cropping systems worldwide has remained on high levels. Our study revealed that neonicotinoid-resistant Q-type strains from Almeria were often more than 100-fold less susceptible to thiamethoxam, acetamiprid and imidacloprid when tested in discontinuous systemic laboratory bioassays. The resistance factors were generally 2- to 3-fold lower in leaf-dip bioassays. In addition to the Spanish strains, we obtained two other highly neonicotinoid-cross-resistant B tabaci greenhouse populations, one from Italy (December 1999) and one from Germany (June 2001). A molecular diagnostic analysis revealed that both strains also belong to the (Spanish) subtype Q of the B tabaci species complex. The resistance levels of Q-type whitefly strains derived from Almeria greenhouses in 1999 remained stable for at least two years, even when maintained in the laboratory without any selection pressure. The biochemical mechanisms conferring resistance to neonicotinoids have not yet been elucidated in detail, but synergist studies suggested a possible involvement of microsomal monooxygenases. Furthermore, we checked two Almerian strains of B tabaci isolated in 1998 and 1999 and demonstrated that neonicotinoid resistance is not due to an altered [3H]imidacloprid binding site of nicotinic acetylcholine receptors.

In vivo and in vitro metabolism of fipronil by larvae of the European corn borer Ostrinia nubilalis.

In vivo and in vitro metabolism of [14C]fipronil was examined in a susceptible European corn borer (Ostrinia nubilalis, Hübner) laboratory strain. [14C]Fipronil penetrated the larval integument slowly, with 71.5% of the applied radioactivity recovered from surface rinses 24 h after topical application. Despite this slow penetration, radioactivity was detected in both the excrement and internal organo-soluble fractions. Radioactivity in the internal aqueous fraction and tissue pellet accounted for less than 0.8% of total radioactivity. The in vivo studies suggest that fipronil oxidation to its sulfone metabolite is the major route of metabolic conversion. In vitro studies were performed using subcellular microsomal fractions isolated from European corn borer larval midguts. Cytochrome P450-dependent monooxygenase activity (methoxyresorufin O-demethylase) was consistently observed in midgut preparations, and formation and detection of the sulfone metabolite in the same midgut preparations was also NADPH-dependent and inhibited by piperonyl butoxide. In vitro metabolism results indicate microsomal monooxygenases are responsible for the conversion of fipronil to its sulfone form in the European corn borer.

Fluorometric Microplate Assay to Measure Glutathione S-Transferase Activity in Insects and Mites Using Monochlorobimane

Elevated levels of glutathione S-transferases (GSTs) play a major role as a mechanism of resistance to insecticides and acaricides in resistant pest insects and mites, respectively. Such compounds are either detoxicated directly via phase I metabolism or detoxicated by phase II metabolism of metabolites as formed by microsomal monooxygenases. Here we used monochlorobimane (MCB) as an artificial substrate and glutathione to determine total GST activity in equivalents of single pest insects and spider mites in a sensitive 96-well plate-based assay system by measuring the enzymatic conversion of MCB to its fluorescent bimane–glutathione adduct. The differentiation by their GST activity between several strains of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), with different degrees of resistance to numerous acaricides was more sensitive with MCB compared to the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Compared to an acaricide-susceptible reference strain, one field population of T. urticae showed a more than 10-fold higher GST activity measured with MCB, in contrast to a less than 2-fold higher activity when CDNB was used. Furthermore, we showed that GST activity can be sensitively assessed with MCB in homogenates of pest insects such as Heliothis virescens, Spodoptera frugiperda (Lepidoptera: Noctuidae), Plutella xylostella (Lepidoptera: Yponomeutidae), and Myzus persicae (Hemiptera: Aphididae).

Regioselective metabolism of taxoids by human CYP3A4 and 2C8: structure-activity relationship.

Paclitaxel and docetaxel are metabolized by liver microsomal monooxygenases into inactive metabolites further eliminated from the body via the bile route. In spite of their close chemical structure, the two drugs are oxidized by two different enzymes; CYP2C8 catalyzes the 6-hydroxylation on the taxane ring of paclitaxel, whereas CYP3A4 oxidizes docetaxel on the tert-butyl group of the lateral chain in C13. Since paclitaxel and docetaxel differ only by two substitutions, the role of individual modifications was investigated; the regioselectivity of hydroxylation was assessed by high-pressure liquid chromatography/mass spectrometry, and enzymes implicated in individual reactions were identified using human liver microsomes and recombinant P450 expressed in Ad293 cells. The biotransformation of docetaxel, 10-deacetylpaclitaxel, and 10-deacetylbaccatin III was steadily increased (2- to 5-fold) by the addition of an acetyl group in position 10, suggesting that the presence of a hydrophobic group in position 10 stimulated hydroxylation by P450 proteins. The absence of the lateral chain at C13 in baccatin III severely impaired the metabolism supported by CYP3A4. The presence of a tert-butyl group in the lateral chain of docetaxel favored the hydroxylation on the tert-butyl by CYP3A4, whereas the presence of a phenyl group in the lateral chain facilitated the oxidation on the taxane ring by CYP2C8. Collectively, these data strongly suggested that the structure of the lateral chain and the nature of substituent in position 10 play an important role in determining the regioselective oxidation by P450 proteins and modulate the reaction rate by human liver microsomes.