Epoxidase Activity Research Articles

Polychlorinated biphenyl (PCB) effects on hepatic mixed function oxidases and porphyria in birds—II. American kestrel

1. In order to study the effects of polychlorinated biphenyls (PCBs) on American kestrels ( Falco sparverius), groups of 6 female birds were fed toxic equivalent doses, estimated from rat studies, of either 2,3,3′4,4′-PCB (3 mg/kg/day) or 3,3′,4,4′,5-PCB (0.05 mg/kg/day) and a comparable dose of 2,2′,4,4′,5,5′-HxCB (4 mg/kg/day) for 4 weeks. 2. Livers were assayed for ethoxyresorufin O-deethylase (EROD), aminopyrine N-demethylase (APND) and aldrin epoxidase (AE) activity and also analyzed for concentrations of porphyrins by HPLC; abdominal fat was analyzed for residues of PCB congeners by GC/ECD. 3. Chronic dosing with 3,3′,4,4′,5-PCB (congener 126) caused significant induction of hepatic EROD and AE; dosing with 2,3,3′4,4′-PCB (congener 105) caused significant hepatic APND induction; dosing with 2,2′,4,4′,5,5′-HxCB (congener 153) caused significant induction of APND and AE. 4. None of the congeners had a significant effect on mean liver weight or mean hepatic porphyrin levels. 5. The data indicate that the relative response of kestrels to dosing with PCB congeners is different from both quail and rats.

Microsomal enzymes in lambs and adult sheep, and their possible relationship to alveld

The difference in susceptibility to alveld between lambs and adult sheep may be caused by differences in the microsomal enzyme activities in their livers. There was no difference in NADPH-cytochrome c reductase or 7-ethoxyresorufin-O-deethylase (EROD) activity between ewes, control lambs and phenobarbitone-dosed lambs 3 weeks after dosing ceased. However, aldrin epoxidase activity was at that time significantly highest in the phenobarbitone-dosed lambs and significantly lowest in the ewes. The liver cytosolic glutathione transferase activity was significantly highest in the ewes and significantly lowest in the control lambs at the same time.

Induction of DNA damage by N-nitrosodiethylamine in rat hepatoma cells: correlation with cytochrome P450-mediated aldrin epoxidase activity

The genotoxic potency of the hepatocarcinogen N-nitrosodiethylamine (NDEA) was tested in various Reuber hepatoma cell lines, which differ in their expression of differentiated liver specific functions including hepatic cytochrome P450 forms, and in rat hepatocytes, with the aim of characterizing the enzymes involved in activation. DNA single-strand breaks assessed by alkaline elution served as an indicator of genetic damage. Aldrin epoxidase activity was used as a marker for various hepatic cytochrome P450 forms. The poorly differentiated cell lines RH35 and H4IIEC3/T were apparently not affected by NDEA; moderate effects were observed in the well-differentiated lines H4IIEC3/G- and 2sFou, and major effects in two other well-differentiated lines, Fao and C2Rev7, and in hepatocytes. The degree of DNA damage in the cell lines correlated positively with the expression of aldrin epoxidase. Furthermore, DNA damage induced by N-nitrosodimethylamine (NDMA) was determined in C2Rev7 cells and in rat hepatocytes in order to assess a possible involvement of the NDMA-metabolizing cytochrome P45oIIE1 in the activation of NDEA by comparing the genotoxic potencies of the two compounds. NDMA was distinctly less effective than NDEA in C2Rev7 cells at all concentrations tested. In hepatocytes, NDMA induced more DNA damage than NDEA at low concentrations, but was slightly less active at high concentrations. The results suggest that NDEA is preferentially metabolized to genotoxic products by one or several cytochrome P450 forms different from P450IIE1.

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.

Regulation of squalene epoxidase activity in rat liver.

Regulation of squalene epoxidase activity was examined in rat hepatic microsomes. The hepatic squalene epoxidase activity was high in the dark period and low in the light period. Three percent cholestyramine feeding increased the hepatic squalene epoxidase activity by 2.5-fold, and the administration of lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, increased its activity by 2.1-fold. Co-administration of cholestyramine and lovastatin had a synergistic effect increasing the activity by 7.0-fold. On the other hand, cholesterol feeding reduced hepatic squalene epoxidase activity to 18%. The addition of sodium fluoride, a phosphatase inhibitor, and the treatment of the microsomes with ATP-Mg2+ had no effect on enzyme activity. HMG-CoA reductase activity has been reported to be regulated by cholesterol and unidentified metabolite(s) derived from mevalonate. However, since squalene epoxidase is less responsive than the reductase to the treatment with lovastatin, squalene epoxidase is thought to be regulated only by cholesterol pool.

Phase I and phase II xenobiotic biotransformation in cultures and co-cultures of adult rat hepatocytes

The aim of this study was to measure the activity of phase I and II key enzymes in the biotransformation of xenobiotics and their inducibility by phenobarbital (2 mM) in two currently used in vitro models, namely adult rat hepatocytes, conventionally cultured or co-cultured with rat epithelial cells derived from primitive biliary duct cells. For phase I, the cytochrome P450 content and the enzymic activities of 7-ethoxycoumarin O-deethylase and aldrin epoxidase have been determined, for phase II glutathione S-transferase activity was measured. In conventional cultures, all phase I parameters investigated declined continuously as a function of culture time. Two mM phenobarbital had inducing effects on 7-ethoxycoumarin O-deethylase and glutathione S-transferases but not on aldrin epoxidase. In co-cultures, after an initial decrease, a steady state situation developed for all the parameters measured, lasting for at least 10 days. The cytochrome P450 content, the 7-ethoxycoumarin O-deethylase, aldrin epoxidase and glutathione S-transferase activities were maintained from 3 to 4 days on at 25, 100, 15 and 50%, respectively, of their corresponding value obtained for freshly isolated hepatocytes. After phenobarbital treatment, the parameters mentioned were significantly increased with the exception of the aldrin epoxidase activity of which the inducibility was nearly completely lost.

Tellurium‐induced neuropathy: A model for reversible reductions in myelin protein gene expression

Inclusion of 1.1% tellurium in the diet of developing rats causes a highly synchronous primary demyelination of peripheral nerves, which is followed closely by a period of rapid remyelination. The demyelination is related to the inhibition of squalene epoxidase activity, which results in a block in cholesterol synthesis and accumulation of squalene. We now report that the demyelination resulting from this limiting of the supply of an intrinsic component of myelin (cholesterol) leads to repression of the expression of mRNA for myelin-specific proteins. Tellurium exposure resulted in an increase in total RNA (largely rRNA) in sciatic nerve, which could not be accounted for by cellular proliferation; these increased levels of rRNA may be a reactive response of Schwann cells to toxic insult and may relate to the higher levels of protein synthesis required during remyelination. In contrast, steady-state levels of mRNA, determined by Northern blot analysis, for P0 and myelin basic protein were markedly decreased (levels after 5 days of tellurium exposure were only 10-15% of control levels as a fraction of total RNA and 25-35% of control levels when the increased levels of total RNA were taken into account). Message levels increased during the subsequent period of remyelination and reached near-normal levels 30 days after beginning tellurium exposure. Although message levels for the myelin-associated glycoprotein showed a similar temporal pattern, levels did not decrease as greatly and subsequently increased sooner than did levels for P0 and myelin basic protein. The coordinate alterations in message levels for myelin proteins indicate that Schwann cells can down-regulate and then up-regulate the synthesis of myelin in response to alterations in the supply of membrane components.

Genetic analysis of aflatoxin B1 activation in rat hepatoma cells

We present a strategy to elucidate the rate-limiting steps in activation of carcinogenic compounds by cytochromes P450. The principle was to select Reuber rat hepatoma cells for resistance to a procarcinogen. The hypothesis was that resistant cells should be systematically deficient in the P450 enzyme(s) involved in the activation process. Here we present an example of the use of this approach using aflatoxin B1 (AFB1), a potent hepatocarcinogen, as the selective agent. Parental cells as well as individual and pooled colonies selected for AFB1 resistance from three independent rat hepatoma lines were characterized for their content of 1) mRNA hybridizing to cDNA and/or oligonucleotide probes for cytochromes P450IIB1, P450IIB2 and albumin; and 2) aldrin epoxidase activity. Parental aflatoxin B1-sensitive cells were shown to express P450IIB1 but not P450IIB2. The majority of the aflatoxin B1-resistant clones failed to accumulate cytochrome P450IIB1 mRNA and expressed no or only very low aldrin epoxidase activity. Albumin mRNA levels remained unchanged, demonstrating that loss of expression of cytochrome P450IIB1 was not a consequence of a general dedifferentiation event. A revertant population showing restoration of both cytochrome P450IIB1 mRNA accumulation and aldrin epoxidase activity was fully sensitive to aflatoxin B1. The correlation between expression of cytochrome P450IIB1 and sensitivity to aflatoxin B1 in both parental cells and revertants strongly suggests that cytochrome P450IIB1 is a major contributor to the activation of aflatoxin B1 in rat hepatoma cells. The kind of strategy described here could be applied to other compounds that become cytotoxic for hepatoma cells following activation by cytochromes P450.

Mechanism of resistance to terbinafine in two isolates of Ustilago maydis

Terbinafine, an antimycotic of the allylamine class, strongly inhibited growth ( I 50 = 0.019 μg/ml) and ergosterol biosynthesis ( I 50 = 0.005 μg/ml) in wild-type (WT) Ustilago maydis sporidia by inhibiting squalene epoxidase activity in the sterol biosynthetic pathway. Two laboratory isolates resistant to terbinafine were characterized. AR217 had I 50 s of 0.930 μg/ml for growth and 0.158 μg/ml for ergosterol synthesis. AR212 had I 50 s of 0.135 μg/ml for growth and 0.028 μg/ml for ergosterol synthesis. Assays of squalene epoxidase activity in cell-free extracts showed a 50% inhibition of total product formation (2,3-oxidosqualene, lanosterol, and 2,3;22,23-dioxidosqualene) at 0.5 μg/ml terbinafine for the WT, whereas AR217 had only a 10% inhibition at this concentration. The basis for resistance in this mutant appears to be a target site change in the enzyme. AR212 showed a similar percentage inhibition as the WT for all tested concentrations, but had a constitutively higher enzyme activity (1.03 pmol product/min/mg protein) in the absence of terbinafine than the WT (0.68 pmol product/min/mg protein) which would adequately explain the resistance of this strain.

Mode of action of terbinafine in Ustilago maydis and characterization of resistant mutants

The colony-forming ability of Ustilago maydis was completely inhibited on agar medium containing 2 μg/ml of the allylamine antimycotic, terbinafine. The colony-forming ability was only 6% of that of the untreated control when sporidia (2.4 × 10 6/ml) were grown for 12 hr in liquid medium with 1 μg/ml of the fungicide prior to plating. A marked accumulation of squalene, no net synthesis of ergosterol, and a 96% inhibition of phospholipid fatty acid synthesis occurred in sporidia incubated for 8 hr with 2 μg/ml of the fungicide. The primary action of terbinafine in U. maydis, as in other fungi, appears to be inhibition of squalene epoxidase activity. Among 152 terbinafine-resistant laboratory isolates selected, only AR217 had a growth rate as high as that of the wild type (WT). AR217 and AR477 showed inhibition of growth similar to that of the WT on agar medium containing low concentrations of sterol C-14 demethylation inhibitors, but the mutants were able to form colonies at higher inhibitor concentrations than the WT. AR212 was hypersensitive to these inhibitors, possibly due to an inability to produce 14α-methylfecosterol, an inefficient substitute for ergosterol.

Polychlorinated biphenyl (PCB) effects on hepatic mixed function oxidases and porphyria in birds. I. Japanese quail

1. Experiments were conducted to compare the effects of polychlorinated biphenyls (PCBs) on Japanese quail ( Coturnix c. japonica). 2. Groups of six female quail were fed toxic equivalent doses estimated from rat studies of either 2,3,3'4,4'-TCB (3 mg/kg/day) or 3,3',4,4',5-PCB (0.05 mg/kg/day) and a comparable dose of 2,2',4,4',5,5'- H×CB (4 mg/kg/day) for 2 or 8 weeks. 3. Livers were assayed for ethoxyresorufin o-deethylase (EROD), 4-chlorobiphenyl hydroxylase (4-CBP), aminopyrine n-demethylase (APND) and aldrin epoxidase (AE) activity. 4. The livers were also analyzed for concentations of porphyrins by HPLC and for residues of the PCBs by GC/ECD. 5. Chronic dosing with 2,3,3',4,4'-PCB caused significant porphyrin accumulation and induction of APND, 4-CBP and EROD. 6. Quail dosed with 3,3'4,4',5-PCB for two weeks exhibited mild but significant porphyria, slight EROD and APND induction and significantly lower thymus weight. 7. Dosing with 2,2',4,4',5,5'-HxCB caused induction of APND and EROD. 8. The data show that the hepatic mixed function oxidase enzymes of the quail respond quite differently than those of rats to PCB exposure.

Effects of dimethylsulphoxide on phase I and II biotransformation in cultured rat hepatocytes

Addition of 2% dimethylsulphoxide (DMSO) to the medium has been described to retain biochemical and morphological differentiation of cultured rat hepatocytes. The maintenance of differentiated hepatocytes for a long time with stable phase I and II biotransformation capacities provides an important model for pharmacological and toxicological studies. This work has been undertaken to study the effects of DMSO on phase I and II parameters in cultured hepatocytes. For phase I cytochrome P-450 content, 7-ethoxycoumarin O-deethylase and aldrin epoxidase activities and for phase II glutathione S-transferase (GST) activity and its isoenzyme profile have been measured. In control cells cytochrome P-450 content and the enzymic activities measured declined as a function of culture time. Addition of DMSO partially prevented the loss of all parameters measured. The changes in GST activity were related to changes in its isoenzyme pattern. In control cells subunits 1 and 2 decreased, 3 and 4 increased and de novo expression of 7 was observed. When DMSO was added, the subunit profile better resembled that observed in vivo and the expression of subunit 7 was suppressed. In conclusion DMSO protects cultured hepatocytes from dedifferentiation and stabilizes the cells.

The biochemical basis for the species difference in hepatic microsomal 4-vinylcyclohexene epoxidation between female mice and rats

Mice but not rats are susceptible to 4-vinylcyclohexene (VCH)-induced ovarian toxicity and carcinogenicity. This is due in part to a 4- to 6-fold greater rate of hepatic microsomal bioactivation of VCH to the ovotoxicant VCH-1,2-epoxide. The biochemical basis for this difference was investigated in microsomes using enzyme induction, enzyme inhibition with chloramphenicol or specific inhibitory antibodies, and correlation with marker steroid hydroxylase activities to associate VCH epoxidation with particular cytochrome P450 forms. Testosterone 6 beta- and 15 alpha-hydroxylase activities and VCH epoxidation were decreased in microsomes from chloramphenicol-treated mice, initially suggesting the possible involvement of P450IIIA and P450IIA forms in VCH metabolism. Although both testosterone 6 beta-hydroxylase and VCH epoxidase activities were increased by dexamethasone treatment (P450IIIA inducer), anti-rat P450IIIA IgG inhibited testosterone 6 beta-hydroxylase (68%) but not VCH epoxidase activity. These latter results do not support the involvement of mouse P450IIIA forms in VCH epoxidation. However, results were obtained which indicated that mouse P450IIA forms are involved in VCH epoxidation. In microsomes from untreated female mice VCH epoxidase activity was inhibited 48% by antibodies to mouse P45015 alpha (P450IIA3) at a concentration that inhibited testosterone 15 alpha-hydroxylase activity by 86%. No protein immunochemically related to mouse P45015 alpha was detected in female rat hepatic microsomes. VCH epoxidation by hepatic microsomes was increased in female mice and rats by phenobarbital treatment and was inhibited by approximately one-third by anti-rat-P450IIB1 IgG in microsomes from untreated animals of both species. Furthermore, microsomal VCH epoxidase and testosterone 16 alpha-hydroxylase activities were lower (34%) in female 129/J mice (deficient in constitutive expression of P450IIB forms) than in B6C3F1 mice. These results suggested partial involvement of P450IIB forms in the microsomal epoxidation of VCH. Therefore, P450 forms IIA and IIB account for the majority of VCH bioactivation in female mouse liver, which explains in part the susceptibility of mice to VCH-induced ovarian toxicity and carcinogenicity.

Organic solvents as modifiers of aldrin epoxidase in reconstituted monooxygenase systems and in microsomes

To examine the response of individual cytochrome P-450 species catalysing the epoxidation of aldrin (Wolff T and Guengerich FP, Biochem Pharmacol 36: 2581–2588, 1987), monooxygenase systems reconstituted from these species were assayed in the presence of 5% (v/v) = 0.87 M ethanol. The activity of cytochromes P-450 pb.b and P-450 pb-d , two enzymes inducible by phenobarbital was increased seven-fold. The activity of two other P-450 enzymes purified from these animals was either inhibited by 50%, as observed for cytochrome P-450 pb-c or remained unchanged, as noted with cytochrome P-450 pcn-e . Two P-450 enzymes purified from untreated rats, cytochromes P-450 ut-f and P-450 ut-h , showed an inhibition by 50 and 20%, respectively, while the activity of cytochrome P-450 ut-a was slightly increased by 50%. Indirect evidence that solvents enhance aldrin epoxidation by interacting with the hemoprotein was obtained by the finding that ethanol stimulated the activity of cytochrome P-450 pb-b already, before addition of the lipid component, l-α-1,2-dilauroyl-in-glycero-3-phosphocholine. The K m of cytochrome P-450 pb.b for NADPH cytochrome P-450 reductase was not altered by ethanol indicating that the interaction between the two enzymes was not affected by the solvent. Other results indicate that the stimulatory solvent binds to a site, apart from the type I or type II binding site. The potency of various hydrophylic solvents to modify aldrin epoxidase activity was assayed in microsomes of rats pretreated with phenobarbital and of untreated male rats. Ethanol, n-propranol, n-butanol, acetone and tetrahydrofuran enhanced enzyme activity of phenobarbital pretreated rats to a maximal extent of two-fold and, at similar concentrations, inhibited the enzyme activity of untreated rats by 50%. The potency of these solvents correlated with their lipophilicity. Methanol and dimethylsulfoxide only slightly modified the activity of induced and noninduced animals. In the presence of 0.5 M n-propranol as the modifying agent, microsomal epoxidase activity of rats pretreated with pregnenolone-16α-carbonitrile, dexamethasone, 3-methylcholanthrene and of control rats was inhibited by 60–80%, whereas the activity of animals pretreated with phenobarbital, DDT, or the polychlorinated biphenyl mixture, Clophen A 50, was stimulated between two- and three-fold. The results reveal that organic solvents frequently used to dissolve monooxygenase substrates may considerably modify the activity of cytochrome P-450 dependent reactions, in particular when purified enzymes are assayed.

Regulation of squalene synthetase and squalene epoxidase activities in Saccharomyces cerevisiae.

Squalene synthetase (EC 2.5.1.21) and squalene epoxidase (EC 1.14 99.7) activities have been measured in cell-free extracts of wild type yeast grown in aerobic and semi-anaerobic conditions as well as in sterol-auxotrophic mutant strains grown aerobically. The results show that both enzymes are induced resulting in an almost two- to five-fold increase in enzymatic activities in mutant strains containing limited sterol amounts and are repressed in the wild type strain cultured in anaerobiosis in excess of sterol. The results show also that squalene epoxidase is repressed by lanosterol, and that the mevalonic acid pool may regulate squalene synthetase levels. The large change in the activities of the two enzymes, depending on the sterol needs of the cells, as well as their low specific activities in comparison with those of the enzymes involved in the early stages of sterol synthesis strongly suggests that squalene synthetase and squalene epoxidase are of importance in regulating the amount of sterol synthesized by yeast.

Effect of ofurace, oxadixyl, and alachlor on xenobiotic biotransformation in the rat liver.

Ofurace, oxadixyl, and alachlor were studied for their effect on hepatic xenobiotic biotransformation in male rats by dosing i.p. with 1 or 100 mg/kg of chemical for 7 days. Ofurace decreased ethoxyresorufin-O-deethylase and aniline p-hydroxylase activities but induced ethoxycoumarin-O-deethylase activity. Glutathione S-transferase and aminopyrine N-demethylase activities responded in a non dose-dependent manner, while cytochrome P-450 content and aldrin epoxidase activities were unchanged. Oxadixyl induced P-450 content, and ethoxycoumarin-O-deethylase and aminopyrine N-demethylase activities. It left ethoxyresorufin-O-deethylase, aldrin epoxidase and epoxide hydrolase activities unchanged and decreased aniline p-hydroxylase activities. Alachlor induced all activities excepting aldrin epoxidase (no change) and aniline p-hydroxylase (decreased). The data indicate that each of these acylanilide pesticides interacts with the monooxygenase system but with differing patterns.

Vitamin K-dependent carboxylase: Partial purification of the enzyme by antibody affinity techniques

The vitamin K-dependent carboxylase activity of bovine liver microsomes has been purified 500-fold by adsorption to an antiprothrombin column and elution with a dodeca peptide which competes with a prothrombin precursor enzyme recognition site. The purified enzyme is devoid of bound precursors, and has the same ratio of vitamin K epoxidase activity to carboxylase activity as the crude microsomal preparation.

Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamic acid residues to form gamma-carboxyglutamic acid residues within the vitamin K-dependent proteins. This enzyme recognizes the gamma-carboxylation recognition site on the propeptide of the precursor forms of the vitamin K-dependent blood coagulation proteins. To purify this enzyme to homogeneity, the carboxylase from bovine liver microsomes was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), the protein was fractionated with ammonium sulfate, and then the enzyme was isolated by affinity chromatography using a synthetic peptide based upon the structure of the prothrombin propeptide. Elution with 10 mM propeptide yielded a single major band on SDS gel electrophoresis with a molecular weight of 77,000. In the presence of high concentrations of propeptide, only minimal carboxylase activity was measurable. Antibodies to the protein inhibited the carboxylase activity in crude preparations. In an alternative affinity purification strategy the propeptide was coupled through an NH2-terminal cysteine to an activated thiol-Sepharose column. The carboxylase-propeptide complex was eluted at 25 degrees C by reductive cleavage of the enzyme-propeptide complex in the presence of detergent and phospholipids. The eluted protein (Mr, 77,000) contained both stable vitamin K-dependent carboxylase and vitamin K epoxidase activity. The protein, purified by either method, was detected as a single band (Mr, 77,000) in a Western blot using anti-carboxylase antibodies. A 10,000-fold purification of carboxylase activity from crude microsomes was estimated. Purified bovine liver vitamin K-dependent carboxylase should facilitate the study of its structure and of the mechanism of action of vitamin K as a cofactor in the reaction catalyzed by this enzyme.

Squalene analogues containing isopropylidene mimics as potential inhibitors of pig liver squalene epoxidase and oxidosqualene cyclase.

Several squalene analogues containing 1,1-dihaloalkene, acetylene, allene, diene, and cyclopropane functionalities were synthesized and evaluated as potential inhibitors of pig liver squalene epoxidase and oxidosqualene cyclase. Both monofunctionalized and bisfunctionalized analogues were prepared. Poor inhibition of squalene epoxidase and oxidosqualene cyclase was found for most compounds (IC50 much greater than 400 microM), with the exception of the monofunctionalized alkynol (IC50 = 300 microM). This alkynol showed mixed-function inhibition with KI = 0.95 mM. Oxidation of the alcohol to the alkynone resulted in loss of epoxidase activity, indicating that the hydroxyl group is necessary for inhibition and that the alkynol is not a proinhibitor. Molecular mechanics calculations indicated that a good inhibitor should possess hydrophobic substituents on an unpolarized, unsaturated system; additionally, the presence of a pro-C-3 hydroxyl group can confer inhibitory potency.

Teflubenzuron resistance and microsomal monooxygenases in larvae of the diamondback moth

Microsomal monooxygenase activities toward a number of model substrates, i.e., 7-methoxy- and 7-ethoxyresorufin and 7-ethoxycoumarin, were from 7- to 28-fold increased in a teflubenzuron-resistant field strain (resistance ratio RR = 31) and in two laboratory-selected teflubenzuron-resistant strains (RR = 18 and 36) of the diamondback moth as compared with a susceptible strain of this insect pest of cruciferous crops. No 7-pentoxyresorufin O-depentylase activity was detected in any of the strains. Aldrin epoxidase activity varied up to 3-fold among the strains studied. Because there is no cross-resistance from pyrethroid resistance to teflubenzuron and because pyrethroid resistance has been attributed to increased microsomal monooxygenase activity as well, we propose that different forms of cytochrome P450s are involved in the detoxication of pyrethroids and teflubenzuron in diamondback moth larvae.