Suicide inactivation of cytochrome P450 by midchain and terminal acetylenes. A mechanistic study of inactivation of a plant lauric acid omega-hydroxylase

Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid omega-hydroxylase (omega-LAH), with [1-(14)C]11-dodecynoic acid (11-DDYA) generates a major metabolite characterized as 1,12-dodecandioic acid. In addition to time- and concentration-dependent inactivation of lauric acid and 11-DDYA oxidation, irreversible binding of 11-DDYA (200 pmol of 11-DDYA bound/mg of microsomal protein) at a saturating concentration of 11-DDYA was observed. SDS-polyacrylamide gel electrophoresis analysis showed that 30% of the label was associated with several protein bands of about 53 kDa. The presence of beta-mercaptoethanol in the incubate reduces 1,12-dodecandioic acid formation and leads to a polar metabolite resulting from the interaction of oxidized 11-DDYA with the nucleophile. Although the alkylation of proteins was reduced, the lauric acid omega-hydroxylase activity was not restored, suggesting an active site-directed inactivation mechanism. Similar results were obtained when reconstituted mixtures of cytochrome P450 from family CYP4A from rabbit liver were incubated with 11-DDYA. In contrast, both 11- and 10-DDYA resulted in covalent labeling of the cytochrome P450 2B4 protein and irreversible inhibition of activity. These results demonstrate that acetylenic analogues of substrate are efficient mechanism-based inhibitors and that a correlation between the position of the acetylenic bond in the inhibitor and the regiochemistry of cytochromes P450 oxygenation is essential for enzyme inactivation.     

Crystal structure of the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability

The in vivo and in vitro effects of the insecticide deltamethrin (DM) on hepatic cytochrome P450 (Cyt P450) monooxygenase were examined in adult carp. The in vivo experiments were carried out with 0.2 microgram/l DM at 20 degrees C. The changes in the hepatic microsomal Cyt P450 content and the Cyt P450-dependent monooxygenase activities were studied in DM-treated fish. Although there were no changes in the Cyt P450 content during the exposure time, after treatment for 24 h all the investigated isoenzyme activities (para-nitrophenetole-O-deethylase, p-NPOD; aminopyrene-N-demethylase, APND; ethylmorphine-N-demethylase, EMND; 7-ethoxycoumarin-O-deethylase, ECOD; and ethoxyresorufin-O-deethylase, EROD) were significantly inhibited. After 72 h, all the activities were still lower than in the control animals. In vitro incubation of liver microsomes with DM led to a concentration-dependent decrease in total microsomal Cyt P450 content. A complete loss of Cyt P450 occurred after a 5-min incubation with 60 microM DM. The maximum in the difference spectra of microsomes was shifted to higher wavelength, showing the strong interaction of DM with Cyt P450. EROD and ECOD activities were inhibited by DM. The in vitro kinetic results on ECOD revealed that the inhibition was of non-competitive type, with K1 = 9.8 +/- 2.3 microM. This study indicates important biochemical effects of DM in fish liver, and suggests that exposure to DM may cause loss of the Cyt P450-dependent metabolism in fish.     

Low carbon monoxide affinity allene oxide synthase is the predominant cytochrome P450 in many plant tissues

A cytochrome P450 with low affinity (2.9 x 10(3) M-1) for CO appears to be the major microsomal P450 in some plant tissues. The presence of low CO affinity cytochrome P450 correlates with its lack of NADPH reducibility and with the presence of high levels of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate peroxidase activity. This activity and low CO affinity are retained by purified tulip cytochrome P450, which appears to be catalytically identical to a flaxseed-derived fatty acid allene oxide synthase P450 described previously [Song, W.-C., & Brash, A.R. (1991) Science 253, 781-784]. Other heme-binding ligands, such as CN- and imidazoles, bind weakly to the allene oxide synthase P450s, suggesting that axial coordination in the heme distal pocket may be hindered. We conclude that low CO affinity is characteristic of the allene oxide synthase P450s and that these P450s constitute a major portion of the microsomal P450 in a variety of plant tissues, particularly from monocot species.     

MP1 encodes an abundant and highly antigenic cell wall mannoprotein in the pathogenic fungus Penicillium marneffei

We characterized the inhibitory activity of several acetylenic and olefinic compounds on cytochrome P450 (CYP)-derived arachidonic acid omega-hydroxylation and epoxidation using rat renal cortical microsomes and recombinant CYP proteins. Among the acetylenic compounds, 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) and N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide were found to be potent and selective inhibitors of microsomal epoxidation with IC50 values of 9 and 13 microM, respectively. On the other hand, 17-octadecynoic acid inhibited both omega-hydroxylation and epoxidation of arachidonic acid with IC50 values of 7 and 5 microM, respectively. The olefinic compounds N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS) and 12, 12-dibromododec-11-enoic acid (DBDD) exhibited a high degree of selectivity inhibiting microsomal omega-hydroxylation with an IC50 value of 2 microM, whereas the IC50 values for epoxidation were 60 and 51 microM for DDMS and DBDD, respectively. Studies using recombinant rat CYP4A isoforms showed that PPOH caused a concentration-dependent inhibition of omega-hydroxylation and 11, 12-epoxidation by CYP4A3 or CYP4A2 but had no effect on CYP4A1-catalyzed omega-hydroxylase activity. On the other hand, DDMS inhibited both CYP4A1- and CYP4A3- or CYP4A2-catalyzed arachidonic acid oxidations. Inhibition of microsomal activity by PPOH, but not DDMS, was time- and NADPH-dependent, a result characteristic of a mechanism-based irreversible inhibitor. These studies provide information useful for evaluating the role of the CYP-derived arachidonic acid metabolites in the regulation of renal function and blood pressure.     

Genetically engineered cells stably expressing cytochrome P450 and their application to mutagen assays

Genetically engineered cells transiently and stably expressing cytochrome P450 (P450), a key enzyme for biotransformation of a wide variety of compounds, have provided new tools for investigation of P450 functions such as P450-mediated metabolic activation of chemicals. This review will focus on the development of mammalian cell lines stably expressing P450s and application to toxicology testings. Stable expression systems have an advantage over transient ones in that a series of the process from metabolic activation of test compounds to the appearance of toxicological consequences occurs entirely in the same intact cells. Indeed, many cell lines stably expressing a single form of mammalian P450 have been established so far and applied to cytotoxic or genotoxic assays, the endpoints of which contained mutations at hprt and other gene loci, chromosomal aberrations, sister chromatid exchanges, micronuclei, morphological transformation, and 32P-postlabeling. Analyses of metabolites of toxic substances have also been carried out, using the intact cells or microsomal fractions prepared from the cells. The stable expression systems clearly indicate the form of P450 enzyme capable of activating a certain chemical. More recently, coexpression of P450 together with other components of microsomal electron transfer systems such as NADPH-cytochrome P450 reductase has been successfully performed to increase the metabolic capacity of the heterologously expressed P450. In addition, to reconstruct the entire metabolic activation system for certain heterocyclic amines, cell lines which simultaneously express a form of human P450 and a phase II enzyme, N-acetyltransferase, were established. These cells were highly sensitive to some carcinogenic heterocyclic amines. In genetic toxicology, such a coexpression system for two or more enzymes will provide useful materials which mimic in vivo activation systems.     

Metabolic activation of aromatic amines by human pancreas

Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas. Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens. Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethyl-amine N-demethylation (P450 2E1); lauric acid omega-hydroxylation (P450 4A1); and 4-(methylnitrosamino)-1-(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alpha-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were <1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [3H]N-hydroxy-ABP) of pancreatic cytosols was high, about twothirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using 32P-post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.     

Flavonoid-induced alterations in cytochrome P450-dependent biotransformation of the organophosphorus insecticide parathion in the mouse

The majority of insecticides currently in use throughout the world belong to the class of the organophosphorus insecticides. Many of these compounds, such as the phosphorothioate insecticides, exert their mammalian toxicity only after undergoing metabolic activation by a variety of cytochrome P450 isoforms to produce their corresponding oxygen analogs (or oxons), which are potent inhibitors of the critical enzyme acetylcholinesterase. Of the many chemicals identified that can modulate cytochrome P450-dependent activities, the flavonoids represent some of the most unusual compounds in that they have been reported to both inhibit and stimulate certain activities. The present study was undertaken to determine if representative flavonoids (at in vitro concentrations of 1-100 microM) can alter the mammalian cytochrome P450-dependent biotransformation and acute toxicity of the phosphorothioate insecticide parathion. The flavonoids 5,6-benzoflavone, flavone, and quercetin had the biphasic effect of stimulating mouse hepatic microsomal parathion oxidation at a concentration of 1 microM, and inhibiting this same activity when increased to 100 microM. In contrast, 7,8-benzoflavone was only inhibitory at all concentrations examined. All the flavonoids examined except quercetin altered the ratio of activation/detoxification of parathion by mouse hepatic microsomes, but had no effect on this same ratio with human CYP1A2. These data suggest that the changes in the activation/detoxification ratio observed with mouse hepatic microsomes resulted from selective inhibition or stimulation of various cytochrome P450 isoforms rather than a flavonoid-induced alteration in the nonenzymatic rearrangement of the putative phosphooxythirane intermediate generated by cytochromes P450 from parathion. Surprisingly, however, none of the four flavonoids in the current study affected the lethality of parathion in vivo, suggesting that the flavonoid-induced alterations in cytochrome P-450-dependent metabolism of parathion documented in vitro were simply not great enough to be of any significance in vivo.     

Characterization of cytochrome P450TYR, a multifunctional haem-thiolate N-hydroxylase involved in the biosynthesis of the cyanogenic glucoside dhurrin

The haem-thiolate N-hydroxylase cytochrome P450TYR involved in the biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin in Sorghum bicolor had recently been isolated. Reconstitution of enzyme activity by insertion of cytochrome P450TYR and NADPH-cytochrome P450-reductase into L-alpha-dilauroylphosphatidylcholine micelles and using tyrosine as substrate results in the formation of p-hydroxyphenylacetaldehyde oxime. Quantitative substrate binding spectra demonstrate that tyrosine and N-hydroxytyrosine are mutually exclusive substrates that bind to the same active site of cytochrome P450TYR. The multifunctionality of cytochrome P450TYR has been confirmed in reconstitution experiments using recombinant cytochrome P450TYR expressed in Escherichia coli. It was earlier reported that an in vitro microsomal system catalyzing all but the last step in the biosynthetic pathway for cyanogenic glucosides exhibits catalytic facilitation (channelling). This observation is explained by the multifunctionality of cytochrome P450TYR. The cytochrome P450TYR sequence represents the first amino acid sequence of a functionally characterized cytochrome P-450 enzyme from a monocotyledonous plant and the first sequence of an N-hydroxylase with high substrate specificity. Multifunctional N-hydroxylases of the cytochrome P-450 type have not previously been reported in living organisms.     

Effects of spironolactone, canrenone and canrenoate-K on cytochrome P450, and 11beta- and 18-hydroxylation in bovine and human adrenal cortical mitochondria

Effects of spironolactone, canrenone and canrenoate-K on adrenal cytochrome P450 (P450) and corticosteroid biosynthesis were examined by studying difference spectra, P450 reduction and corticoid hydroxylation in mitochondrial preparations isolated from zona fasciculata and zona glomerulosa of bovine adrenals and from adrenal adenoma and hyperplastic adrenal cortex removed from patients with hyperaldosteronism. All three agents bound to P450 producing type I difference spectra and underwent hydroxylation. They all inhibited 11beta-hydroxylation in bovine adrenal at 30 muM and higher concentrations. Canrenone, the most potent inhibitor, blocked enzyme activity by 60% at a concentration of 60 muM. Spironolactone stimulated P450 reduction. The order of potency of inhibition was found to correlate with the order of affinity of these agents for P450. 11beta-Hydroxylase in human adrenal appeared to be less sensitive to canrenone. All three agents or their hydroxylated metabolites blocked 18-hydroxylation in bovine adrenal at lower concentrations. Canrenoate-K, being the most effective, inhibited 52% at 20 muM. Low concentrations of canrenone (2.5-5.0 muM) were without effect on 11beta-hydroxylase but markedly inhibited 18-hydroxylation (62-76%) in hyperplastic human adrenals. The inhibitors produced mixed type inhibition of 11beta-hydroxylation and competitive type inhibition of 18-hydroxylation. These findings indicate that at low concentrations spironolactone and its major metabolites, canrenone and canrenoate-K, or their hydroxylated metabolites, can directly interfere with the biosynthesis of aldosterone in bovine and certain human adrenal cortical tissue.     

Inhibition of [3H]quinpirole binding by a monoamine oxidase inhibitor in subcellular fractions of rat striatum

[3H]Quinpirole is a dopamine agonist with high affinity for D2-like dopamine receptors. A number of non-dopaminergic compounds, most notably monoamine oxidase inhibitors (MAOIs), inhibit the binding of [3H]quinpirole, but not other D2-like agonists and antagonists, in rat striatal membranes by a mechanism that does not involve the enzymatic activity of MAO. To further characterize this novel interaction, the subcellular distribution of spiperone-displaceable, "D2-like" [3H]quinpirole-labeled sites in rat striatum was assessed and compared with the distribution of MAOI-displaceable [3H]quinpirole binding (MQB). "D2-like" [3H]quinpirole binding exhibited similar nanomolar affinity in the crude synaptosomal (P2), crude microsomal (P3), and ribosomal, post-microsomal (P4) fractions. Total binding activity (fmol bound/fraction) of "D2-like" [3H]quinpirole binding was concentrated in the synaptosomal fraction (P2B). The subcellular distribution of MQB paralleled that of "D2-like" [3H]quinpirole binding. This suggests that "D2-like" [3H]quinpirole binding and MQB occur at a common membrane-bound binding site.     

Microsomal cytochrome P450 dependent oxidation of N-hydroxyguanidines, amidoximes, and ketoximes: mechanism of the oxidative cleavage of their C=N(OH) bond with formation of nitrogen oxides

Oxidation by rat liver microsomes of 13 compounds involving a C=N(OH) function (including N-hydroxyguanidines, amidoximes, ketoximes, and aldoximes) was found to occur with the release of nitrogen oxides such as NO, NO2-, and NO3-. The greatest activities were observed with liver microsomes from dexamethasone-treated rats (up to 8 nmol of NO2- nmol of P450(-)1 min-1). A detailed study of the microsomal oxidation of some of these compounds was performed. Oxidation of N-(4-chlorophenyl)-N'-hydroxy-guanidine led to the formation of the corresponding urea and cyanamide in addition to NO, NO2-, and NO3-. Formation of all these products was dependent on NADPH, O2, and cytochromes P450. Oxidation of two arylamidoximes was found to occur with formation of the corresponding amides and nitriles in addition to nitrogen oxides. Oxidation of 4-(chlorophenyl)methyl ketone oxime gave the corresponding ketone and nitroalkane as well as NO, NO2-, and NO3-. These reactions were also dependent on cytochromes P450 and required NADPH and O2. Mechanistic experiments showed that microsomal oxidations of amidoximes to the corresponding nitriles and of ketoximes to the corresponding nitroalkanes are not inhibited by superoxide dismutase (SOD) and are performed by a cytochrome P450 active species, presumably the high-valent P450-iron-oxo complex. On the contrary, microsomal oxidation of N-hydroxyguanidines to the corresponding cyanamides was greatly inhibited by SOD and appeared to be mainly due to O2*- derived from the oxidase function of cytochromes P450. Similarly, microsomal oxidations of N-hydroxyguanidines and amidoximes to the corresponding ureas and amides were also found to be mainly performed by O2*-, as shown by the great inhibitory effect of SOD (70-100%) and the ability of the xanthine-xanthine oxidase system to give similar oxidation products. However, it is noteworthy that other species, such as the P450 Fe(II)-O2 complex, are also involved, to a minor extent, in the SOD-insensitive microsomal oxidative cleavages of compounds containing a C=N(OH) bond. Our results suggest a general mechanism for such oxidative cleavages of C=N(OH) bonds with formation of nitrogen oxides by cytochromes P450 and NO-synthases, with the involvement of O2*- and its Fe(III) complex [(FeIII-O2-) or (FeII-O2)] as main active species.     

Differential interaction of erythromycin with cytochromes P450 3A1/2 in the endoplasmic reticulum: a CO flash photolysis study

The kinetics of CO binding to cytochromes P450, measured by the flash photolysis technique, were used to probe the interaction of erythromycin with cytochromes P450 in rat liver microsomes. Addition of erythromycin generates substrate difference spectra using microsomes from rats treated with phenobarbital or dexamethasone but not from untreated rats, showing that it binds to P450s induced by these agents. In contrast, erythromycin and/or a monoclonal antibody to P450 3A1/2 accelerated CO binding to microsomes from rats treated with phenobarbital but had no effect on microsomes from untreated or dexamethasone-treated rats. Based on the differential amounts and inducibilities of the P450 3A1 and 3A2 forms in these microsomal samples, these results indicate that erythromycin increased the rate for P450 3A2 but not P450 3A1. The divergent effects of erythromycin on these P450s, which exhibit 89% sequence similarity, were consistent with a model of the P450 substrate binding site in which erythromycin forms a more rigid complex with P450 3A1 than P450 3A2. These results demonstrate the sensitivity of P450 conformation/dynamics to substrate binding, and show that CO binding kinetics can distinguish among closely related P450s in a microsomal environment.     

Messenger RNA synthesis, transport, and storage in silkmoth ovarian follicles

The effect on liver microsomal enzyme activity of three steroid contraceptive drug (SCD) combinations was compared in rats, mice and guinea-pigs. Lynestrenol plus mestranol, norethisterone plus mestranol and norethynodrel plus mestranol were given orally for 4 consecutive days (acute treatment) or 30 days (chronic treatment) at various doses eliciting an experimentally controlled antifertility activity which varied in its extent. In rats and mice all the combined treatments (with the exception of norethynodrel plus mestranol in mice) were active as inducers of liver microsomal enzymes. This induction seems to be mediated mainly by the progestogenic compounds. Oestrogens showed a very poor effect bordering on significance only in a few cases. No effect on liver microsomal protein or cytochrome P 450 concentration was obtained after treatment with doses capable of increasing the microsomal enzyme activity. The activity of the liver microsomal enzymes did not appear to be reduced immediately (2 h) after the last administration of the SCD given during 4 or 30 days. Contraceptive treatments at doses capable of eliciting complete antifertility activity were inactive on liver microsomal enzyme activity in guinea-pigs.     

Change in activity of microsomal cytochrome P-450-dependent monooxygenases from rat liver exposed to chronic low doses of radiation

The activity changes of cytochrome P-450-dependent monooxygenases of hepatic microsomal fraction were studied in, experimental animals after prolonged application (1 month) of low radiation doses (0.258 mkl/kg). The obtained results show the increase in catalytic activities of main forms of cytochrome P-450, participating in steroid hydroxylation, as well as the decrease in total content of cytochrome P-450 in hepatic microsomal fraction and the lowering of its demethylase activity.     

Spectral analysis and catalytic activities of the microsomal mixed-function oxidase system of the sea anemone (phylum: Cnidaria)

Cnidarians are primarily marine organisms with a wide and diverse habitat worldwide. Previous studies from our laboratory demonstrated the presence of proteins in the molecular mass range of 50-60 KDa that were recognized by several antibodies raised against fish cytochromes P450 of the CYP 1, 2, and 3 families in microsomes prepared from the columnar regions of 5 species of sea anemones (Heffernan et al. Mar Environ Res 1996;42:353-357). Pursuant to those studies we report herein results of spectral analyses, NAD(P)H-oxidoreductase and ethoxyresorufin O-dealkylase (EROD) of sea anemone microsomal fractions. The predominant feature in the difference spectrum of dithionite (DTN)-reduced, CO-liganded anemone microsomes was a peak with lambda max of approximately 418 nm, which slowly increased for about 20 min and decreased after about 40 min. A relatively lower amplitude 450 nm peak was attained within 5 min of CO addition and was stable for up to 90 min. The 450 nm peak did not increase concomitant to the decrease in the 418 nm peak suggesting that the latter is not denatured P450. A significantly larger 450 nm peak was attained in CO-difference spectra when DTN was added prior to CO. NADPH-dependent cytochrome c (P450) reductase of the sea anemones was 1.8-3.9 nmol/min/mg protein, which is at the lower end of the range observed in invertebrates. NADH-cytochrome c reductase was 9-25 nmol/min/mg protein, while the NADH-ferricyanide (b5) reductase ranged from 73-232 nmol/min/mg protein. When microsomal EROD activity was measured under conditions in which the reaction was linear with respect to protein concentration, a decrease in fluorescence was typically observed for the initial 15 min of the time course and then increased linearly for up to 90 min; initial velocities were determined from the increasing linear region. NADPH was the preferred cofactor for EROD activity and the NAD(P)H-EROD activity was higher in Anthopleura elegantissima than Anthopleura xanthogrammica. The Bunodosoma cavernata NADPH-EROD activity was barely noted at the detection limit of the assay and NADH-EROD activity was not detected. These results are consistent with a functional P450-dependent MFO system in cnidarians, with characteristics both similar to and unique from other marine invertebrates.     

Preparation of an activity-inhibiting monoclonal antibody against human placental aromatase cytochrome P450

We produced a murine monoclonal antibody (MAb) to human placental aromatase cytochrome P450. This MAb, designated MAb3-2C2, was selected on its ability to suppress aromatase activity. The specificity of this MAb was assessed by selective immunoprecipitation of 125I-labeled aromatase cytochrome P450 as well as by the identification of a 55-kDa protein, which was enriched and purified by immunoaffinity chromatography on a MAb-coupled Sepharose 4B column. The MAb was able to suppress both human placental and ovarian microsomal aromatase. Species differences of aromatase were recognized by MAb3-2C2 on the basis of differential immunosuppression of aromatase activity. The antibody had no effect on non-aromatase cytochrome P450s. MAb3-2C2 gave negative results with human placental aromatase P450 in the Western blot analysis. The data presented indicate that MAb3-2C2 is specific for aromatase cytochrome P450 and that its epitope is located in a fragile tertiary conformation of the enzyme, thus making it capable of sensitively affecting catalysis.     

Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II

In renal proximal tubule epithelial cells, a membrane-associated phospholipase A2 (PLA2) is a major signaling pathway linked to angiotensin II (Ang II) type 2 receptor (AT2). The current studies were designed to test the hypothesis that membrane-associated PLA2-induced release of arachidonic acid (AA) and/or its metabolites may serve as an upstream mediator of Ang II-induced mitogen-activated protein kinase (MAPK) activation. Ang II stimulated transient dose-dependent phosphorylation of MAPK with a maximum at 1 microM (10 min). Inhibition of PLA2 by mepacrine diminished both AA release and MAPK phosphorylation, induced by Ang II. Furthermore, AA itself induced time- and dose-dependent phosphorylation of MAPK, supporting the importance of PLA2 as a mediator of Ang II signaling. The effects of both Ang II and AA on MAPK phosphorylation were protein kinase C independent and abolished by the inhibitor of cytochrome P450 isoenzyme, ketoconazole. Moreover, 5,6-epoxyeicosatrienoic acid and 14,15-epoxyeicosatrienoic acid, the cytochrome P450-dependent metabolites of AA, significantly stimulated MAPK activity in renal proximal tubule epithelial cells. These observations document a mechanism of Ang II-induced MAPK phosphorylation, mediated by PLA2-dependent release of AA and cytochrome P450-dependent production of epoxy derivatives of AA.     

Effect of dietary oils, cholesterol and antioxidant vitamin supplementation on liver microsomal fluidity and xenobiotic-metabolizing enzymes in rats

This study was undertaken to compare the effects of four oils: corn (C), olive (O), hazelnut (H) or fish (F), and the intake of two supplements: cholesterol, 1% (Ch) or dl-alpha-tocopherol acetate, 500 mg/kg, and beta-carotene, 30 mg/kg (V), on liver microsomal fluidity, cyt P450 content and aniline hydroxylase (AH), aminopyrine-N-dimethylase (AND) and UDP-glucuronyltransferase (UDP-GT) activities. Male Sprague-Dawley rats (n = 6/group) were fed semipurified diets containing 15% oil, without or with Ch or V, for 20 days. Dietary intake and feed efficiency were lower in rats fed F. Relative liver weight was higher in animals fed F, similar in O and H, and lower in the group fed C. The intake of V increased feed intake in C+V group and decreased the relative liver weight of F+V group, which also decreased with the intake of F+Ch. Ch intake increased the relative liver weight in all groups consuming vegetable oils. Cyt P450 content was higher in rats fed F. Decreased cyt P450 content was observed in C+Ch and F+Ch groups, while it augmented in H+Ch group. Mixture V increased cyt P450 in rats fed C+V, F+V and O+V. The highest membrane fluidity was observed in rats fed F. Fluidity was also higher in group H versus O or C. The intake of Ch decreased microsomal fluidity in all groups, while V induced an increase in microsomal fluidity in group O+V. Rats fed F exhibited higher enzyme activities. AND activity increased with V only in rats fed H+V, while AH activity increased with V intake in groups F+V and O+V. In the C+V group, fluidity was not affected by V, while the cyt P450 content and UDP-GT activity increased. The O+V group exhibited lower UDP-GT activity and higher fluidity and cyt P450 content. The activity of AH decreased in groups F+Ch and C+Ch. UDP-GT activity was higher in rats fed F. It diminished after the intake of Ch in H+Ch and F+Ch. These results indicate that although AH and AND act in the same microsomal metabolic pathway, their localization into the membrane may be determinant of their activity and the response to dietary lipids. It is shown that F intake exerts the most significant effects upon liver microsomal properties, e.g. higher fluidity, cyt P450 content and enzymatic activities, an effect that prevails over the intake of the supplements tested.     

Androgen receptor dependent and independent activities of testosterone on hepatic microsomal drug metabolism

Administration of testosterone for 6 days to intact female and castrate male BALB/cJ mice stimulated hepatic microsomal ethylmorphine N-demethylase activity and cytochrome P-450 content by 50-75%. Testosterone also stimulated hepatic microsomal NADPH-oxidase activity, but to a lesser degree. To probe the mechanism of this effect of androgens, two antiandrogens (cyproterone acetate and flutamide) were employed. Since cyproterone acetate was a potent stimulator of hepatic microsomal ethylmorphine N-demethylase activity and cytochrome P-450 content, no antiandrogenic activity of this steroid could be detected. By contrast, flutamide alone had little effect on either ethylmorphine N-demethylase activity or cytochrome P-450 content. However, this drug effectively blocked the stimulatory effects of testosterone on ethylmorphine N-demethylase activity and cytochrome P-450 content but not on NADPH-oxidase activity. This effect was not species specific, since flutamide also prevented androgen stimulation of ethylmorphine metabolism in adult castrate and prepubertal male Fisher rats. The testosterone-induced increase of hepatic weight and microsomal protein content was not affected by the administration of flutamide. The observations are consistent with the hypothesis that androgens have two distinct effects on the liver. First, testosterone may act as a general, nonspecific stimulant of liver weight and microsomal protein content which is independent of the androgen receptor. Secondly, testosterone action in the liver may be expressed via an androgen-specific or androgen receptor-dependent mechanism which controls, in part, the cytochrome P-450-dependent demethylase system.     

Investigation on possible antioxidative properties of the NMDA-receptor antagonists ketamine, memantine, and amantadine in comparison to nicanartine in vitro

Possible antioxidative properties of three N-methyl-D-aspartate (NMDA)-receptor antagonists, the anesthetic ketamine and the antiparkinson drugs memantine and amantadine were investigated in vitro on the microsomal cytochrome P450 (P450) system of rat livers and on rat whole blood chemiluminescence in comparison to nicanartine, a substance with known antiatherosclerotic, hypolipemic and antioxidative capacity. For this purpose, the effects on NADPH- and iron-stimulated lipid peroxidation (LPO), hydrogen peroxide (H2O2) production, and NADPH- and iron-stimulated lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) were examined using rat liver microsomes. Additionally, the influence on LM amplified whole blood chemiluminescence after zymosan activation of polymorphonuclear leukocytes (WB-CL) was investigated. Furthermore, binding to P450 and effects on P450 mediated monooxygenase function, as measured by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and ethylmorphine N-demethylation (END), were assessed. Nicanartine concentration dependently reduced LPO and H2O2 production already at a concentration of 1 microM, whereas LC and LM amplified CL and WB-CL were not affected. EROD and END were concentration dependently diminished starting at 1 microM, and ECOD already at 0.1 microM. Ketamine decreased LPO, H2O2 production and LM and LC amplified CL, starting at 100 microM. WB-CL was significantly diminished already at 10 microM. EROD and ECOD were inhibited at 10 and 100 microM and END at 100 microM. With memantine a concentration dependent inhibition of LPO and WB-CL was seen at 100 and 1000 microM and a reduction of LC and LM amplified CL only at 1000 microM. H2O2 production was not affected. EROD and ECOD were significantly diminished by a concentration of 100 microM. No effect was observed on END. Amantadine significantly reduced LPO and WB-CL, but only at 1000 microM. H2O2 production and LC and LM amplified CL were not affected. EROD was significantly diminished at 100 microM, whereas no influence was seen on ECOD and END. Nicanartine displayed type II or reverse type I, ketamine, memantine and amantadine type I substrate binding to P450. The highest binding affinity to P450 was seen with nicanartine, followed by ketamine, memantine and then amantadine. These results demonstrate, that all four substances seem to act as radical scavengers and/or as inhibitors of the oxidative function of P450. All four substances seem to interfere with the monooxygenase function of P450. This may result in a possible influence on the biotransformation of endogenous as well as of foreign compounds. The effects of nicanartine were much more pronounced than those of ketamine, memantine, and amantadine.