Presence Of Metyrapone Research Articles

Metabolic response in liver and Brockmann bodies of rainbow trout to inhibition of lipolysis; possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis.

We previously demonstrated in rainbow trout that the decrease in circulating levels of fatty acid (FA) induced by treating fish with SDZ WAG 994 (SDZ) induced a counter-regulatory response in which the activation of the hypothalamus-pituitary-interrenal (HPI, equivalent to mammalian hypothalamus-pituitary-adrenal) axis was likely involved. This activation, probably not related to the control of food intake through FA sensor systems but to the modulation of lipolysis in peripheral tissues, liver and Brockmann bodies (BB, the main site of pancreatic endocrine cells in fish), would target the restoration of FA levels in plasma. To assess this hypothesis, we lowered circulating FA levels by treating fish with SDZ alone, or SDZ in the presence of metyrapone (an inhibitor of cortisol synthesis). In liver, the changes observed were not compatible with a direct FA-sensing response but with a stress response, which allows us to suggest that the detection of a FA decrease in the hypothalamus elicits a counter-regulatory response in liver, resulting in an activation of lipolysis to restore FA levels in plasma. The activation of these metabolic changes in liver could be attributable to the activation of the HPI axis and/or to the action of sympathetic pathways. In contrast, in BB, changes in circulating FA levels induce changes in several parameters compatible with the function of FA-sensing systems informing about the decrease in circulating FA levels.

Counter-regulatory response to a fall in circulating fatty acid levels in rainbow trout. Possible involvement of the hypothalamus-pituitary-interrenal axis.

We hypothesize that a decrease in circulating levels of fatty acid (FA) in rainbow trout Oncorhynchus mykiss would result in the inhibition of putative hypothalamic FA sensing systems with concomitant changes in the expression of orexigenic and anorexigenic factors ultimately leading to a stimulation of food intake. To assess this hypothesis, we lowered circulating FA levels treating fish with SDZ WAG 994 (SDZ), a selective A1 adenosine receptor agonist that inhibits lipolysis. In additional groups, we also evaluated if the presence of intralipid was able to counteract changes induced by SDZ treatment, and the possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis by treating fish with SDZ in the presence of metyrapone, which decreases cortisol synthesis in fish. The decrease in circulating levels of FA in rainbow trout induced a clear increase in food intake that was associated with the decrease of the anorexigenic potential in hypothalamus (decreased POMC-A1 and CART mRNA abundance), and with changes in several parameters related to putative FA-sensing mechanisms in hypothalamus. Intralipid treatment counteracted these changes. SDZ treatment also induced increased cortisol levels and the activation of different components of the HPI axis whereas these changes disappeared in the presence of intralipid or metyrapone. These results suggest that the HPI axis is involved in a counter-regulatory response in rainbow trout to restore FA levels in plasma.

Bupropion Hydroxylation as a Selective Marker of Rat CYP2B1 Catalytic Activity

Benzyloxyresorufin-O-dealkylation (BROD) is usually used as a marker of cytochrome P450 (P450) 2B1 in rat. However, some reports show that CYP1A2 is also highly implicated. The purpose of the present study was to establish bupropion (BUP) hydroxylation, but not BROD, as a selective in vitro marker of CYP2B1 catalytic activity. IC(50) for BROD and BUP hydroxylation were equivalent (40.8 ± 4.6 and 41.8 ± 3.4 μM, respectively) when using liver microsomes from β-naphthoflavone-pretreated rats in the presence of metyrapone (CYP2B1 inhibitor). When using the same microsomes in the presence of CYP1A1/2-selective inhibitor α-naphthoflavone, we found an IC(50) of 2.5 × 10(-3) ± 0.8 × 10(-3) μM for BROD and >100 μM for BUP hydroxylation. These results suggest that CYP2B1 is similarly involved in both activities, whereas CYP1A2 is involved in BROD activity but not in BUP hydroxylation. BUP hydroxylation was assessed in microsomes from baculovirus-infected insect cells coexpressing NADPH-P450 oxidoreductase, and 14 rat P450s and kinetic parameters (K(m) and V(max)) were determined. BUP hydroxylation was predominantly catalyzed by CYP2B1 (75% of total hydroxybupropion formation), low activity was detected with CYP2E1 and CYP2C11 (10.9 and 8.7% of total hydroxybupropion, respectively), and activity was almost undetectable with the other P450 isoforms at saturating substrate concentrations (2500 μM), thereby validating the use of BUP as a diagnostic in vitro marker of CYP2B1 catalytic activity in rat.

Characterization of the binding of [3H]‐clobenpropit to histamine H3‐receptors in guinea‐pig cerebral cortex membranes

1 We have investigated the binding of a novel histamine H3-receptor antagonist radioligand, [3H]- clobenpropit ([3H]-VUF9153), to guinea-pig cerebral cortex membranes. 2 Saturation isotherms for [3H]-clobenpropit appeared biphasic. Scatchard plots were curvilinear and Hill plot slopes were significantly less than unity (0.63+/-0.03; n = 12+/-s.e.mean). The radioligand appeared to label two sites in guinea-pig cerebral cortex membranes with apparent affinities (pKD') of 10.91+/-0.12 (Bmax = 5.34+/-0.85 fmol mg(-1) original wet weight) and 9.17+/-0.16 (Bmax = 23.20+/-6.70 fmol mg(-1)). 3 In the presence of metyrapone (3 mM) or sodium chloride (100 mM), [3H]-clobenpropit appeared to label a homogeneous receptor population (Bmax=3.41+/-0.46 fmol mg-1 and 3.49+/-0.44 fmol mg(-1), pKD' = 10.59+/-0.17 and 10.77+/-0.02, respectively). Scatchard plots were linear and Hill slopes were not significantly different from unity (0.91+/-0.04 and 0.99+/-0.02, respectively). Granisetron (1 microM), rilmenidine (3 microM), idazoxan (0.3 microM), pentazocine (3 microM) and 1,3-di-(2-tolyl)guanidine (0.3 microM) had no effect on the binding of [3H]-clobenpropit. 4 The specific binding of [3H]-clobenpropit appeared to reach equilibrium after 25 min at 21+/-3 degrees C and remained constant for >180 min. The estimated pKD' (10.27+/-0.27; n = 3+/-s.e.mean) was not significantly different from that estimated by saturation analysis in the presence of metyrapone. 5 A series of histamine H3-receptor ligands expressed affinity values for sites labelled with [3H]-clobenpropit which were not significantly different from those estimated when [3H]-R-alpha-MH was used to label histamine H3-receptors in guinea-pig cerebral cortex membranes.

In situ 1H NMR study of cytochrome P450 inhibition by metyrapol in mycobacterium aurum MO1

In situ 1H NMR was used to monitor incubations of Mycobacterium aurum MO1 with thiomorpholine or morpholine in the presence of metyrapone, a specific inhibitor of P450. Unexpected signals were detected and assigned to metyrapol, the reduction product of metyrapone. The inhibitory effects of racemic metyrapol and of its (-)-enantiomer were compared to that of metyrapone. They are similar.

Disruption of endogenous regulator homeostasis underlies the mechanism of rat CYP1A1 mRNA induction by metyrapone.

The transcriptional induction of the cytochrome P-450 1A1 (CYP1A1) gene by xenobiotics such as polyaromatic hydrocarbons is dependent on their interaction with the aryl hydrocarbon receptor. Administration of the structurally unrelated compounds metyrapone (a cytochrome P-450 inhibitor) or dexamethasone (a glucocorticoid) to male rats does not induce hepatic CYP1A1 mRNA. However, administration of both metyrapone and dexamethasone to male rats results in the induction of hepatic CYP1A1 mRNA expression. The induction response is mimicked in vitro in cultured rat hepatocytes by the addition of metyrapone and dexamethasone to a serum-free culture medium, suggesting that these compounds act directly on the liver in vivo to effect hepatic CYP1A1 mRNA induction. An examination of the characteristics of CYP1A1 induction by metyrapone and dexamethasone in combination in vitro indicate that at least 6 h of treatment is required for detectable levels of CYP1A1 mRNA to accumulate in hepatocytes. In contrast, beta-naphthoflavone, which is known to bind to the aryl hydrocarbon receptor to effect CYP1A1 gene expression, induces detectable levels of CYP1A1 mRNA within 2 h of treatment. CYP1A1 mRNA is also induced when hepatocytes are treated with metyrapone in combination with the protein synthesis inhibitor cycloheximide but not with dexamethasone in combination with cycloheximide, indicating that CYP1A1 mRNA induction is strictly dependent on the presence of metyrapone and suggesting that the metyrapone-associated induction of CYP1A1 mRNA is dependent on a loss of a constitutively expressed protein that functions to suppress CYP1A1 gene expression. The role of dexamethasone in metyrapone-associated induction of CYP1A1 is probably mediated through the glucocorticoid receptor since the glucocorticoid receptor antagonist RU486 reduces the levels of CYP1A1 mRNA induced by metyrapone and dexamethasone in combination. Increasing the levels of the photosensitizer riboflavin present in the culture medium 10-fold and exposure to light increases the levels of CYP1A1 mRNA induced by metyrapone and dexamethasone in combination in vitro, suggesting that photoactivation of inducing medium constituent(s) might be required for induction. Failure to induce CYP1A1 mRNA by co-administration of metyrapone and dexamethasone in hepatocytes cultured in a balanced salt solution with or without photoactivation indicates that induction is dependent on a photoactivated component of the culture medium and not on metyrapone or dexamethasone alone. The addition of tryptophan in the presence of riboflavin to the balanced salt solution restores CYP1A1 mRNA induction by metyrapone alone and induction is increased when medium is exposed to light, indicating that induction is dependent on tryptophan photoactivation in vitro. Metyrapone failed to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin for specific binding to the aryl hydrocarbon receptor in rat liver cytosolic fractions. These results suggest that CYP1A1 might be induced in rats by metyrapone through an indirect mechanism associated with an elevation in the level of an endogenously generated inducer such as photoactivated product(s) of tryptophan and not because of metyrapone's interacting with the aryl hydrocarbon receptor. The dependence of CYP1A1 induction on dexamethasone or cycloheximide suggests that derepression by a glucocorticoid receptor-modulated negative-acting factor of CYP1A1 gene expression might be critical to induction by metyrapone.

Metyrapone is a competitive inhibitor of 11beta-hydroxysteroid dehydrogenase type 1 reductase.

The present study was designed to examine the effects of metyrapone in vitro on the activities of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) types 1 and 2, the two intracellular enzymes responsible for the metabolism of glucocorticoids. Enzymatic activities of 11beta-HSD1 and 2 were determined by a radiometric conversion assay using cortisol and cortisone as physiological substrates. The enzyme activity assays were carried out in the absence and presence of metyrapone using sheep liver and kidney microsomes as the source of 11beta-HSD1 and 2, respectively. It was found that metyrapone inhibited the reductase activity of 11beta-HSD1 in a dose-dependent manner with an apparent Ki of 30 microM. Moreover, this inhibition was competitive because the Km for cortisone was increased in the presence of metyrapone. In contrast, metyrapone showed biphasic effects on the dehydrogenase activity of 11beta-HSD1, in that it increased the activity at concentrations lower than 100 microM but decreased it at higher concentrations. However, under similar conditions, metyrapone had little effect on the unidirectional dehydrogenase activity of 11beta-HSD2. In conclusion, the present results provide the first direct evidence that metyrapone is a competitive inhibitor of 11beta-HSD1 reductase, and that it also exerts biphasic effects on 11beta-HSD1 dehydrogenase activity. These findings indicate that metyrapone influences peripheral glucocorticoid metabolism through its regulation of 11beta-HSD1 activity, in addition to its classic inhibitory effects on adrenal steroid biosynthesis. It is therefore imperative that this novel extra-adrenal effect of metyrapone be considered when this drug is used in the diagnosis and treatment of adrenocorticoid-related diseases.

Effects of metyrapone on expression of CYPs 2C11, 3A2, and other 3A genes in rat hepatocytes cultured on matrigel

Hepatocytes cultured on matrigel express many liver-specific functions, but the levels and activities of the predominant male-specific rat hepatic CYPs, 3A2 and 2C11, decline rapidly in culture. Metyrapone maintains the level of total cytochrome P450 of rat hepatocytes in primary culture, but the mechanism underlying this effect has not been completely elucidated. The present study sought to determine whether metyrapone acts solely to stabilise CYP proteins in rat hepatocytes cultured on matrigel, or whether it also influences mRNA levels of the encoding genes. Metyrapone maintained the level of total cytochrome P450 in cultured hepatocytes so that values were > 200% of those found in untreated control cells 24 hr after isolation. At this time, CYP3A2-mediated testosterone 6β-hydroxylation was approximately 7-fold higher in hepatocytes cultured in the presence of metyrapone than in control cells, and CYP2C11-dependent testosterone 2α- and 16α-hydroxylation activities were between 2 and 3-fold greater. The results inferred from catalytic activities were supported by immunoquantitation of CYP3A and 2C11 proteins. The trend of increased CYP protein levels in metyrapone-treated cells continued throughout the 48-hr culture period. In control cells, CYP3A2 and 2C11 mRNA levels fell abruptly in culture to reach values at 24 hr that were < 30% of those in freshly isolated cells; addition of metyrapone failed to arrest this fall. However, treatment of cells with metyrapone considerably elevated levels of one or more CYP3A subfamily mRNA species, as detected by a riboprobe based on the cDNA for CYP3A1 (‘CYP3A1-like mRNA’) that were demonstrated, by another riboprobe, not to be CYP3A2 or RNCYP3AM. RT-PCR of mRNA prepared from cultured hepatocytes, followed by restriction mapping of the cloned cDNAs was used to characterise the CYP3A induced by metyrapone. This revealed that elevated levels of the CYP3A1-like mRNA were attributable to induction of RL33/cDEX mRNA; there were no CYP3A1 cDNAs isolated from these cells. These data are interpreted as indicating that metyrapone stabilises the expression of cytochrome P450 in culture by both pre- and posttranslational mechanisms. The particular mechanism employed is gene-specific, whereby even the highly homologous genes CYP3A2, RL33/cDEX and, possibly, RNCYP3AM are subject to different types of regulation in the presence of metyrapone.

Ketoconazole inhibits oxidative modification of low density lipoprotein

Known cytochrome P450-dependent oxygenase inhibitor ketoconazole (5–50 μM) blocked the murine macrophagemediated modification of human low density lipoprotein (LDL) as measured by production of thiobarbituric acid-reactive substance, stimulation of [ 125I]LDL degradation in a fresh set of macrophages and LDL electrophoretic mobility, in a dose-dependent manner with complete inhibition at 30–40 μM. When resident macrophages were incubated with LDL in the presence of metyrapone, methoxsalen and α-naphthaflavone at concentrations that have been shown to inhibit the cytochrome P450-dependent oxygenases, there was no change in LDL modification. Induction of benzo[α]pyrene hydroxylase activity in macrophages by 24 h incubation with benzo[α]pyrene was accompanied by a 1.5-fold increase of LDL modification which has been leveled down by ketoconazole as well as methoxsalen and α-naphthaflavone. Furthermore, ketoconazole effectively diminished cell-free LDL oxidation induced by iron, but not copper ions, and reduced the spontaneous and zymosan-stimulated lucigenin-amplified chemiluminescence of macrophages. The data allow us to suggest that ketoconazole inhibits LDL oxidation by acting as an iron chelator and/or inhibitor of prooxidant forms of iron-containing enzymes.

Production of 26-deoxymonensins A and B by Streptomyces cinnamonensis in the presence of Metyrapone

Metyrapone, a potent cytochrome P-450 inhibitor, added at 9 mM to a submerged culture of Streptomyces cinnamonensis caused partial inhibition of total monensin biosynthesis and coproduction of new metabolites, 26-deoxymonensins A and B. The latter was isolated as its 25-O-methyl derivative. Metyrapone was simultaneously reduced to metyrapol. All of these compounds were identified by nuclear magnetic resonance spectroscopy and mass spectrometry.

The effect of metyrapone on cytokinin ([8- 14C]Benzylaminopurine) metabolism in mature green tomato pericarp

Cytokinin, [8-14C]Benzylaminopurine, metabolism in tomato pericarp was followed during a 3 h period utilizing thin layer chromatography and visualization by fluorography. Fluorography indicated the formation of at least 7 metabolites during 3 h. Cytokinin metabolism was reduced by approximately 40% in 3 h by the presence of 250 microM metyrapone, an inhibitor of cytochrome P-450 related enzyme systems. In the presence of metyrapone, the number of radioactive metabolites on the thin layer plate was reduced from 7 to 4 and 2 of these were unique to the metyrapone-treated sample. These data suggest the initial step in benzylaminopurine metabolism in tomato pericarp may be mediated by a cytochrome P-450 related enzyme system which is altered in the presence of metyrapone.

11-Deoxycortisol induces hepatic tryptophan oxygenase in rats.

11-Deoxycortisol (cortexolone) has previously been used as a glucocorticoid antagonist in vitro and in adrenalectomized rats. Antiglucocorticoid properties of 11-deoxycortisol in intact rats were examined by studying the effect of 11-deoxycortisol on the induction of hepatic tryptophan oxygenase (TO) by corticosterone. No antiglucocorticoid effect was observed. When 11-deoxycortisol was injected into rats, the TO activity increased. This was probably mainly caused by an elevation of the serum corticosterone level. The induction of TO by 11-deoxycortisol was inhibited by metyrapone. However, 11-deoxycortisol (100 mg/kg) was still not a glucocorticoid antagonist even in presence of metyrapone.

Chemical modification of cytochrome P-450 LM2 : Characterization of tyrosine as axial heme iron ligand trans to thiolate

Phenobarbital-inducible isozyme cytochrome P-450 LM2 (RH, reduced-flavoprotein: oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes has been modified with N-acetylimidazole and tetranitromethane. Up to four tyrosine residues of cytochrome P-450 LM2 are accessible to O-acetylation and to nitration. N-Demethylase activity, spectral dissociation constants and substrate binding kinetics of differently acetylated enzyme indicate the existence of two groups of accessible tyrosines also differing in their reactivity towards N-acetylimidazole. The fast-reacting tyrosine residue representing the first group is involved in the binding of the type II substrate aniline and appears to be located near the heme as shown by the protecting effect of the inhibitor metyrapone against modification, but obviously is not necessary for N-demethylation. Acetylation of one further tyrosine residue, however, caused an almost complete inhibition of the enzyme, indicating its involvement in the catalytic mechanism at the active center. Nitration of two tyrosine residues inactivates to about 20%. Obviously the third and fourt tyrosine residue are without functional importance. The experiments evidencing two functionally linked tyrosines are in line with HPLC analyses of tryptic peptides of cytochrome P-450 LM2 nitrated in the presence of metyrapone which gave evidence for the location of two distinct tyrosine residues in the active center. Nitration of tyrosine residues results in the partial formation of a hyperporphyrin spectrum of cytochrome P-450 LM2. Its appearance is prevented in the presence of metyrapone and can be reversed by reduction of the nitrotyrosinate.

Generation of free radical metabolites and superoxide anion by the calcium indicators arsenazo III, antipyrylazo III, and murexide in rat liver microsomes.

At the concentrations usually employed as a Ca2+ indicator, arsenazo III undergoes a one-electron reduction by rat liver microsomes to produce an azo anion radical as demonstrated by electron spin resonance spectroscopy. Either NADH or NADPH can serve as a source of reducing equivalents for the production of this free radical by rat liver microsomes. The steady state concentration of the azo anion radical is proportional to the square root of the protein concentration, suggesting that the radical decays through a nonenzymatic second order process. The steady state concentration of the azo anion radical is not altered in the presence of metyrapone or CO, and is decreased in the presence of NADP+ or p-hydroxymercuribenzoate. These observations suggest that the formation of arsenazo III anion radical is mediated through NADPH-cytochrome P-450 reductase and not by cytochrome P-450. Under aerobic conditions, addition of arsenazo III to rat liver microsomes produces an increase in electron flow from NAD(P)H to molecular oxygen, generating both superoxide anion and hydrogen peroxide. The steady state concentration of the azo anion radical, but neither oxygen consumption nor superoxide anion formation, is enhanced by calcium and magnesium, suggesting an enhanced azo anion radical-stabilization by complexation with the metal ions. Accordingly, the arsenazo III anion radical signal is abolished in the presence of paramagnetic metal ions (Fe3+, Gd3+, and Ni2+) and enhanced in the presence of other diamagnetic metal ions (La3+). Antipyrylazo III is less effective than arsenazo III in increasing superoxide anion formation by rat liver microsomes, and gives a much weaker ESR spectrum of an azo anion radical. Murexide is reduced to the monodehydro-5,5'-iminobarbituric acid radical by rat liver microsomes, and its efficiency as a superoxide anion generator is intermediate between arsenazo III and antipyrylazo III.

Dynamics of xenobiotic metabolism by isolated rat hepatocytes using a multichannel perifusion system.

The multichannel perifusion system in recirculating and non-recirculating (single-pass) mode has been used to monitor the rate of oxidative metabolism of three model substrates--7-ethoxycoumarin, dichloronitroanisole and aldrin. With control hepatocytes, the rate of de-ethylation of 7-ethoxycoumarin derived from recirculating mode was essentially similar to the rate obtained with conventional flask-incubated cell suspensions. The formation of 7-hydroxycoumarin glucuronide and sulphate by hepatocytes exposed to 7-ethoxycoumarin demonstrated the retention of conjugative ability of cells in the perifusion system. The rate of demethylation of dichloronitroanisole to dichloronitrophenol was low whilst aldrin epoxidation to dieldrin was rapid using control hepatocytes in recirculating mode. The inductive effect of phenobarbitone on hepatic mixed-function oxidases was demonstrated by a marked increase in the rate of 7-ethoxycoumarin (nine-fold) and dichloronitroanisole (64-fold) dealkylation by hepatocytes from phenobarbitone-treated animals in recirculating mode. The rate of substrate oxidation by hepatocytes perifused in the recirculating and the single-pass mode were the same. With dichloronitroanisole as substrate and a single-pass mode, the rate of dichloronitrophenol formation declined rapidly on perifusion with substrate-free medium and rapidly re-attained steady state on re-introduction of the substrate; the presence of metyrapone effectively inhibited dichloronitroanisole metabolism. The perifusion system is recommended for the study of the dynamics of xenobiotic metabolism by isolated mammalian hepatocytes.

Generation of free radicals induced by nifurtimox in mammalian tissues.

Nifurtimox is reduced by rat liver microsomes to a nitro anion-free radical as indicated by ESR spectroscopy. This subcellular fraction gives a steady state radical concentration which is proportional to the square root of the protein concentration, suggesting that the nifurtimox anion radical is a necessary intermediate in the reduction and that the radical decays through a nonenzymatic second order process. The steady state concentration of the anion radical in the microsomal system is not decreased by superoxide dismutase or catalase, thus indicating that neither the superoxide anion nor hydrogen peroxide is an intermediary in the generation of the anion radical. The steady state concentration of the anion radical in the microsomal system is also not altered in the presence of metyrapone or CO and is decreased in the presence of NADP+ and p-chloromercuribenzoate. This observation suggests that the formation of nifurtimox anion radical is mediated through NADPH-cytochrome P-450 (c) reductase and not by the cytochrome P-450 system. In accordance with this interpretation, a model system consisting of NADPH and FMN-reduced nifurtimox to the nitro anion-free radical. Nifurtimox anion radical generation is significantly stimulated by rat brain and testes homogenates. The enhanced free radical formation may be the basic cause of nifurtimox toxicity in mammals.

2-Acetylaminofluorene metabolism in rat liver microsomes: Formation of 9-hydroxy-2-acetylaminofluorene and effect of hepatic enzyme modifiers

9-Hydroxy-2-acetylaminofluorene [ N-(9-hydroxy-9H-fluoren-2-yl)acetamide; 9-OH-2-AAF] was isolated and identified as a metabolite of 2-acetylaminofluorene (2-AAF) in rat liver microsomes. Evidence for this metabolite was provided by thin-layer chromatography (tlc), inverse isotope dilution analysis, pH partition, and nmr spectroscopic studies. 9-Oxo-2-acetylaminofluorene [ N-(9-oxo-9H-fluoren-2-yl)acetamide; 9-OXO-2-AAF] was also detected and identified (by tlc) as a microsomal metabolite of 2-AAF. Microsomal conversion of 2-AAF to 9-OH-2-AAF and 9-OXO-2-AAF was dependent upon the presence of NADPH and was inhibited by a carbon monoxide atmosphere. Increased formation of 9-OH-2-AAF was noted in microsomes obtained from rats treated with the hepatic enzyme modifiers, phenobarbital and pregnenolone-16α-carbonitrile and inhibited in the presence of metyrapone. In contrast, treatment with 3-methylcholanthrene or chlordane did not modify 9-OH-2-AAF or 9-OXO-2-AAF formation. Also, rates of 9-OXO-2-AAF formation were increased only by prior administration of phenobarbital and were unaffected by metyrapone. These data provide evidence for the involvement of specific types of cytochrome P-450 in the 9-carbon hydroxylation reaction of 2-AAF.

Relationship between alveolar PO2 and the rate of p-nitroanisole O-demethylation by the cytochrome P-450 pathway in isolated rabbit lungs.

The relationship between alveolar PO2 and the rate of O-demethylation of p-nitroanisole, a model substrate for cytochrome P-450 -linked mixed-function oxidation, was evaluated in the isolated rabbit lung perfused with Krebs-Ringer bicarbonate buffer. The appearance of the product, p-nitrophenol, in the pulmonary perfusate was measured spectrophotometrically, The PO2 of the ventilating gas was varied with an accurate gas mixing pump and measured with an electrochemical O2 analyzer. In control lungs ventilated with 5% CO2 in air, the rate of p-nitrophenol production was approximately equal to 3.1 +/- 0.04 (mean +/- SE; n = 9) mumol/h per g dry wt. p-Nitrophenol production was unaltered when O2 in the ventilating gas was decreased to 1%, but it was depressed reversibly when alveolar O2 WAS 0.1% OR LESS AND WAS ABOLISHED DURING VENTILATION WITH 0.005% O2. The rate of the reaction was inhibited by 50% when alveolar PO2 was 0.3 mm Hg representing and intracellular [O2] OF approximately equal to muM. In the presence of metyrapone (0.1--1 mM), an inhibitor of cytochrome P-450-dependent reactions, p-nitrophenol production was 0.07--0.17 mumol/h per g dry wt. Ventilation of lungs with varying CO concentration in 20% O2 resulted in 50% inhibition of p-nitrophenol production when CO concentration was 10% (CO/O2 = 0.5). These results indicated that O-demethylation of p-nitroanisole by the lung is a cytochrome P-450-dependent reaction and that its rate is not affected until alveolar PO2 is less than 1 mm Hg.

Sites of metyrapone inhibition of steroid biosynthesis by rat adrenal mitochondria.

ABSTRACT Rat adrenal mitochondrial preparations supplemented with an NADPH-generating system were incubated with various labelled substrates in order to evaluate further the action of metyrapone on the utilization of cholesterol for steroid biosynthesis1). The formation of pregnenolone from [4-14C] cholesterol (0.5, 1.0 and 2.0 μCi) in 5, 10 and 15 min incubations was decreased by 72–82 % in the presence of metyrapone (0.5 mm). Similarly, the generation of labelled side chain fragments from [26-14C]-cholesterol was depressed 36–42 % by 0.2 mm metyrapone and 65–70 % by 1.0 mm inhibitor during 30, 60 and 90 min incubations. Metyrapone inhibition of the side chain cleavage was not observed, however, if cholesterol was replaced as substrate by its C-20 hydroxylated analog: The formation of pregnenolone from [7-3H]20α-hydroxycholesterol (0.5, 1.0 and 2.0 μCi), also an NADPH-mediated mitochondrial reaction, was not affected by similar concentrations of metyrapone, indicating that the inhibition observed with cholesterol as substrate is not related to non-specific toxic effects, to interference with NADPH generation or to impairment of NADPH function in the mitochondrial electron transport system. Parallel incubations with [4-14C] 11-deoxycorticosterone and with [4-14C] cholesterol over a wide range of inhibitor concentrations (0.01–1.0 mm) demonstrated that the effects of metyrapone on 11β-hydroxylation and on the side chain cleavage were dose-related; at low concentrations, however, metyrapone was a more potent inhibitor of 11β-hydroxylation than of cholesterol conversion to pregnenolone. These studies demonstrate clearly in the rat adrenal the dual inhibitory effect of metyrapone sug-gested by previous in vivo and in vitro observations in man.