Hans G. Jonen

Influence of pyridine and some pyridine derivatives on spectral properties of reduced microsomes and on microsomal drug metabolizing activity

Abstract Addition of pyridine, 3-(3-pyridyl)-propanol, reduced metyrapone and some other pyridine derivatives to dithionite reduced rat liver musomes results in the formation of a six banded spectrum with absorption maxima at 425, 446, 527, 539, 555 and 568 nm. With metyrapone as the ligand, a 425 nm band can only be observed during development of the spectrum. The ratio of the two Soret bands depends on the pH. The 446 nm band is maximal at pH 7·8 in phenobarbital stimulated musomes and decreases on both lowering and increasing the pH. For metyrapone the apparent spectral dissociation constant K s for the absorbance change at 446 nm was 16 μM in musomes from untreated animals and was decreased to 2·4 μM after phenobarbital pretreatment. In 3-methylcholantrene stimulated musomes the K s value for metyrapone was increased about 25 fold and amounted to 390 μM. The K s value for reduced metyrapone in unstimulated musomes was similar to that for metyrapone. Less pronounced pretreatment effects were, however, observed with this ligand leading to decreased K s values in both PB and MC stimulated musomes. Pyridine and 3-(3-pyridyl)-propanol were only bound to reduced cytochrome P-450 in millimolar concentrations. All tested ligands were able to inhibit musomal drug metabolism. Metyrapone was the most potent inhibitor of drug demethylation exerting 50 per cent inhibition of p -nitroanisole demethylation at 4 μM and of aminopyrine demethylation at 86 μM. Aniline hydroxylation was inhibited by 50 per cent by millimolar concentrations of all inhibitors at aniline concentrations of 4 mM.

Interruption of the enterohepatic circulation of phenprocoumon by cholestyramine.

The effect of cholestyramine (12 gm/day divided into 3 doses) on the pharmacokinetics and pharmacodynamics of a single intravenous dose (30 mg) of phenprocoumon was studied in 6 normal subjects. Cholestyramine treatment led to an increase in the rate of elimination of phenprocoumon in all. Total clearance increased 1.5‐ to 2‐fold. The total anticoagulant effect per dose was considerably reduced during treatment with cholestyramine. Binding studies in vitro showed that phenprocoumon is strongly bound to cholestyramine and that at a given cholestyramine concentration the percentage of phenprocoumon bound remained constant over a large concentration range of phenprocoumon. The results suggest that phenprocoumon undergoes extensive enterohepatic recycling in man which can be effectively interrupted by cholestyramine.

Enhancement of Microsomal Aniline and Acetanilide Hydroxylation by Haemoglobin

1. Haemogloblin and myoglobin enhance rat liver microsomal p-hydroxylation of aniline and acetanilide. Microsomal N-demethylation of ethylmorphine and aminopyrine is not increased by haemoproteins. 2. The enhancement of microsomal p-hydroxylation is maximal at high substrate concentration and high haeme compound concentration. 3. Detergent-purified NADPH-cytochrome c reductase, free flavins and manganese ions considerably increase the haemoglobin-mediated, tissue-free hydroxylation of aniline. Microsomal aniline hydroxylation is not enhanced by haeme, ferric ion or albumin. 4 Catalase and cyanide ions are powerful inhibitors of haemoglobin-mediated aniline hydroxylation both in the presence and absence of tissue. Carbon monoxide inhibits the hydroxylase activity of the tissue-free system to a smaller extent than that of a system containing microsomes plus haemoglobin whereas p-chloromercuribenzoate inhibits only the flavoprotein-dependent hydroxylation of aniline mediated by haemoglobin. 5. Several possibilities of interactions between substrate, microsomes and haeme compounds are proposed.

Reductive and oxidative metabolism of nitrofurantoin in rat liver

SummaryThe elimination of nitrofurantoin was studied in the isolated rat liver using a recirculating hemoglobin-free perfusion system. The most rapid clearance of nitrofurantoin (0.1 mM) was found under hypoxia (8 ml/min) or anoxia (11 ml/min) indicating a fast and oxygen-sensitive reductive metabolism. The hepatic elimination of nitrofurantoin under anaerobic conditions apparently is not catalyzed by xanthine oxidase, aldehyde oxidase or cytochrome P-450 as judged from the lack of influence of the inhibitors (0.1 mM) allopurinol, menadione, metyrapone, α-naphthoflavone or of carbon monoxide (50%; v/v).Under aerobic conditions the hepatic clearance of nitrofurantoin is rather low (1 ml/min) indicating only a minor role of the liver for the overall elimination of this drug under in vivo conditions. In the presence of ethanol (38 mM) the clearance of nitrofurantoin was accelerated significantly (2 ml/min) suggesting an ethanol-mediated reductive metabolism even under aerobic conditions. The hepatic oxygen uptake was enhanced by nitrofurantoin in a dose-dependent manner and reached 160% of the control rate (1.4 μmol O2×g−1×min−1) at 0.2 mM nitrofurantoin. This effect may indicate the formation of active oxygen species in the intact organ.4-Hydroxylation of nitrofurantoin could only be detected after induction with β-naphthoflavone or 3-methylcholanthrene. The hepatic clearance of nitrofurantoin was raised to 3–4 ml/min after pretreatment with these agents which are known to induce the same group of cytochromes P-450. Hepatic clearance and hydroxylation rates after induction with 3-methylcholanthrene were found to be strongly correlated. Metyrapone (0.1 mM) and α-naphthoflavone (0.1 mM) inhibited the 4-hydroxylation of nitrofurantoin by 90 and 100%, respectively. The aerobic microsomal metabolism of nitrofurantoin was also enhanced by induction with 3-methylcholanthrene. The results indicate that 4-hydroxylation of nitrofurantoin is catalyzed by a 3-methylcholanthrene-inducible form of cytochrome P-450.

Reductive Drug Metabolism in Isolated Perfused Rat Liver under Restricted Oxygen Supply

1. Hepatic azo and nitro reductase activities were studied in the perfused rat liver under normal and restricted oxygen supply. 2. Formation of sulphanilamide or p-aminobenzoic acid from neoprontosil or p-nitrobenzoic acid under aerobic conditions of liver perfusion was negligible, even at a reduced oxygen saturation of a pO2 of 300 mm Hg in the haemoglobinfree perfusion system. At a pO2 of 200 mm Hg reductase activities were almost maximal. 3. Conjugation of sulphanilamide (0-08 mM) was similar under aerobic and anaerobic conditions. Hepatic elimination of p-aminobenzoic acid (0-08 mM) showed an oxygen-dependent increase for 15 min after addition of substrate. 4. p-Nitroanisole demethylation was inhibited 80% under hypoxic perfusion at 200 mm Hg pO2 and was completely inhibited after gassing with anoxic mixtures. 5. Restitution of aerobic conditions after 30 min anaerobic perfusion restored hepatic respiration, lactate pyruvate ratio, and pH value to levels found under aerobic conditions, but bile flow remained 50% reduced.

Effect of phenobarbital and 3-methyl-cholanthrene administration in vivo and isooctane extraction in vitro on metyrapone binding to reduced cytochrome P-450☆

Abstract The changes of metyrapone binding to reduced cyctochrome P-450 during phenobarbital and methylcholanthrene pretreatment were compared with the effect of the inducers on cytochrome P-450 content of rat liver microsomes. The maximal absorbance change at 446 nm caused by addition of metyrapone to reduced microsomal preparations was increased under the influence of both inducers. The apparent spectral dissociation constant Ks was decreased by phenobarbital pretreatment but increased by methylcholanthrene pretreatment. These changes of metyrapone binding also occurred if the increase in cytochrome P-450 content was suppressed by concomitant pretreatment with the inhibitor of protein synthesis cycloheximide. During methylcholanthrene pretreatment the increase of the maximal absorbance at 446 nm which appeaf ed after addition of metyrapone began rapidly and reached a maximum 36 hr after a single dose of the inducer. At this time no increase of cytochrome P-450 content was detected. The changes of metyrapone binding observed after methylcholanthrene pretreatment could be imitated by extraction of the microsomes with isooctane. The inducer mediated modifications of the binding characteristics of metyrapone are discussed in relation to the lipid environment of cytochrome P-450 and to the synthesis of an inducer specific form of the hemoprotein.

Differential inhibition of biphenyl hydroxylation in perfused rat liver

SummaryA differential inhibition of biphenyl hydroxylation by α-naphthoflavone and metyrapone was observed in isolated pefused rat liver. α-Naphthoflavone inhibited 2- and 4-hydroxylation in livers from β-naphthoflavone-pretreated animals but had no effect on both reactions in livers from phenobarbital-pretreated animals. Metyrapone inhibited 2- and 4-hydroxylation in phenobarbital-stimulated livers, but only insignificant inhibition of 2-hydroxylation and a slight enhancement of 4-hydroxylation by metyrapone was observed in β-naphthoflavone-stimulated livers.Conjugation of 2-hydroxybiphenyl and 4-hydroxybiphenyl by isolated perfused livers was also studied. 4-Hydroxybiphenyl preferentially formed sulphates in livers from untreated animals but after induction glucuronidation was as effective as sulphation or even exceeded sulphation. Only glucuronic acid conjugates of 2-hydroxybiphenyl were detected.

Enhancement of Nitro Reduction in Rat Liver Microsomes by Haemin and Haemoproteins

1. Reductive metabolism of p-nitrobenzoic acid and neoprontosil in rat liver microsomes was studied in the presence of haemin, haemoglobin and myoglobin. 2. Microsomal nitro reduction is enhanced 4-fold in the presence of haemoglobin, whereas azo reduction is not affected. 3. Microsomal nitro reduction is enhanced to a similar extent by haemoglobin, haemin and boiled haemoglobin, whereas myoglobin is about half as active. 4. Maximal enhancement of microsomal nitro reductase activity by haemoglobin is achieved at high substrate concentration (6 mM) and low microsomal protein concentration (0.5--1.0 mg/ml). 5. Control microsomal nitro reduction as well as the haemoglobin-enhanced microsomal nitro reduction are inhibited completely by O2 and CO whereas potassium azide as a ligand of ferric haem iron is a less potent inhibitor.

INDUCTION OF CYTOCHROME P-448 BY 3-METHYLCHOLANTHRENE IN THE RAT DURING INHIBITION OF PROTEIN SYNTHESIS IN VIVO *

Administration of cycloheximide in vivo during induction of rats with 3-methylcholanthrene prevents the increase in total cytochrome P-450 content usually seen under the influence of the inducer. The population of cytochromes P-450 in the livers of these animals is, however, similar to that in the completely induced animals. Microsomal aryl hydrocarbon hydroxylase activity and biphenyl-2-hydroxylation are enhanced severalfold and biphenyl-4-hydroxylation is enhanced twofold. Monooxygenase activity shows the same pattern of preferential inhibition as in microsomes from animals which had received the inducer only. The affinity of the reduced cytochromes for the ligand metyrapone is considerably decreased both after treatment with 3-methylcholanthrene and with 3-methylcholanthrene plus cycloheximide. A distinct blue shift of the absorbance maxima of the reduced P-450-CO complex and of the reduced P-450-metyrapone complex occurs after concomitant treatment with the inducer and the inhibitor of protein synthesis. The pattern of cytochrome P-450 bands after SDS polyacrylamide gel electrophoresis of microsomes from animals treated with both drugs is similar to that of MC-stimulated microsomes but different from that of control microsomes. These data show that during inhibition of protein synthesis in vivo 3-methylcholanthrene induces the same pattern of cytochromes P-450 as under normal conditions of induction while the increase in total cytochrome P-450 content is almost completely prevented.

Enzymic control of irreversible binding of metabolically activated benzo(a)pyrene in perfused rat liver by monooxygenase activity.

Addition of [3H]-benzo(a)pyrene to the perfusion medium of isolated rat livers results in irreversible binding of radioactivity to DNA, RNA and protein. Binding to DNA accounted for about 0.1% of the total radioactivity which was bound in livers from animals treated with oil or saline and was increased by a factor of 3–5 after pretreatment of the animals with β-naphthoflavone or with phenobarbital. When the inhibitiors of monooygenase activity, α-naphthoflavone or metyrapone, were present in the perfusion medium, irreversible binding was reduced in livers from both β-naphthoflavone- and phenobarbital-pretreated animals, irrespective of the inhibitor used.In livers from animals treated with oil or saline protein and a RNA fraction containing tightly associated protein were able to bind [3H]-benzo(a)pyrene metabolites to about the same extent but after induction by pretreatment with β-naphthoflavone binding to the RNA fraction was enhanced to a much higher extent than binding to the protein fraction. Pretreatment with phenobarbital did not result in an increased irreversible binding to RNA and protein.A considerable amount of 15–25% of the total radioactivity added to the perfusion medium was excreted into the bile after treatment of the animals with the tested inducers of monooxygenase activity compared to an excretion of 3% in animals treated with oil or saline.The results indicate that nucleic acid and protein adduct formation in the liver is controlled by the action of the cytochrome P-450-dependent monooxygenases.ZusammenfassungNach Zugabe von [3H]-Benzo(a)pyren zum Perfusionsmedium der isolierten Rattenleber wird eine irreversible Bindung von Radioaktivität an DNA, RNA und Protein beobachtet. Die Bindung an DNA betrug in Lebern von öl- bzw. kochsalzbehandelten Tieren etwa 0,1% der gesamten gebundenen RadioaktivitätNach Vorbehandlung der Tiere mit β-Naphthoflavon oder Phenobarbital stieg die Bindung an DNA um den Faktor 3–5 an. In Gegenwart von α-Naphthoflavon oder Metyrapon als Inhibitoren der Monooxygenase wurde die irreversible Bindung sowohl nach β-Naphthoflavon als auch nach Phenobarbitalvorbehandlung geringer.In Lebern von öl- bzw. kochsalzbehandelten Tieren wurde etwa gleich viel [3H]-Benzpyren pro mg an Protein und an die RNA-Fraktion, die noch fest assoziiertes Protein enthielt, gebunden. Nach Vorbehandlung mit dem Induktor β-Naphthoflavon wurde die Bindung an die RNA-Fraktion in erheblich größerem Ausmaß gesteigert als diejenige an die Proteinfraktion, während nach Vorbehandlung mit Phenobarbital die Bindung an RNA und Protein nicht anstieg.Gegenüber einer Ausscheidung in die Galle von 3% der gesamten ins Perfusionsmedium eingesetzten Radioaktivität bei Lebern von öl- bzw. kochsalzbehandelten Tieren stieg die biliäre Elimination nach Vorbehandlung mit den untersuchten Induktoren der Monooxygenase auf 15–25% der eingesetzten Benzpyrenmenge an.Die Ergebnisse sprechen dafür, daß die Bildung von Nucleinsäure- und Proteinaddukten in der Leber durch die Aktivität der Cytochrom P-450-abhängigen Monooxygenasen kontrolliert wird.