Georg F. Kahl

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.

Formation of benzo[a]pyrene metabolite-nucleoside adducts in isolated perfused rat and mouse liver and in mouse lung slices.

Abstract The formation of benzo[a]pyrene metabolite-nucleoside adducts in perfused rat and mouse liver and in mouse lung slices was studied by Sephadex LH20 chromatography. In liver from β-naphthoflavone-pretreated rats, four different deoxyribonucleoside complexes were observed; these are tentatively attributed to DNA modification by the 7,8-diol-9, 10-epoxide(s), secondary metabolites of benzo[a]pyrene quinones, the 4,5-oxide, and secondary metabolites of benzo[a]pyrene phenols. The diol-epoxide-deoxyribonucleoside adduct was also detected in perfused liver and in lung slices from 3-methylcholanthrene-treated genetically responsive C57BL/6N mice, whereas no adducts were detectable in such samples from 3-methylcholan-threne-treated genetically nonresponsive DBA/2N mice. In perfused liver of phenobarbital-pretreated rats, the 4,5-oxide-deoxyribonucleoside adduct was present. These results suggest that some of the benzo[a]pyrene metabolite-nucleoside complexes generated by microsomes and deproteinized DNA in vitro also occur in the intact rodent liver and lung tissues. Furthermore, complexes with the diol-epoxide(s) were observed with RNA from perfused liver of β-naphthoflavone-treated, but not from untreated or phenobarbital-treated rats. Complexes between ribonucleoside(s) and the diol-epoxide(s) were also found in perfusedliver or lung slices from genetically responsive but not from genetically nonresponsive mice.

Effects of synthetic progestagens on drug metabolism in rat liver musomes

Evidence is presented that the gestagenic components of hormonal contraceptives norethisterone acetate d-norgestrel and lynestrenol and the progestagen allylestrenol inhibit the oxidative metabolism of p-nitroanisole and aniline in rat liver moicrosomes at concentrations between 5 X 10 less than -5 greater than M and 5 X 10 less than -4 greater than M. Liver microsomes were prepared from male Sprague-Dawley rats. The animals were injected ip with 80 mg of sodium phenobarbital/kg daily for 3 days. They were sacrificed 24 hours after the last injection and 12 hours after food was withdrawn. Livers were perfused in situ with ice-cold saline excised and homogenized. Nuclear fragments and mitochondria were sedimented by centrifugation of the homogenates. Details of technique for the determination of drug metabolizing activity are given. The p-nitroanisole demethylation and aniline hydroxylation in the phenobarbital-stimulated liver micorsomes were inhibited by norethisterone acetate d-norgestrel lynestrenol and allylestrenol. Inhibition was dose-dependent up to a steroid concentration of 1 X 10 less than -4 greater than if complete solubility is assumed. The progestagens inhibit drug metabolism when the concentration of substrate and inhibitor are similar. The situation of women taking oral contraceptives is different from the conditions of the in vitro experiments. In therapy the dose does not exceed 5 mg which is not sufficient to inhibit metabolism of drugs given at the same time. Others have reported excretion of unmetabolized drugs in users of oral contraceptives. More clinical research is needed on the effect of hormonal contraceptives on drug elimination.

The effect of metyrapone on cellular respiration and microsomal drug oxidation.

Abstract Five mM metyrapone increases the lactate-pyruvate ratio in the perfusion fluid of perfused rat livers by a factor of 9 and in rat liver slices by a factor of 3. Furthermore, it decreases the rate of oxygen uptake of liver slices by 50 per cent. The reduced metabolite of metyrapone also inhibits respiration of liver slices. From these results evidence is taken for an inhibitory effect of metyrapone and its main metabolite on the respiratory chain leading to a change of the redox status of the cell and to an increase of aerobic glycolysis. In mouse liver microsomes the rate of oxygen consumption in the absence of exogenous substrates is not affected by 2 × 10 −4 M metyrapone, although metyrapone in this concentration already inhibits microsomal drug hydroxylation. When the inhibitor SKF 525-A is added oxygen uptake increases, thereby confirming the nature of the inhibitor as a substrate of microsomal hydroxylases. This increase can be diminished by the subsequent addition of 2 × 10 −4 M metyrapone. These results indicate that metyrapone as an inhibitor of drug metabolism acts on the binding of substrates to cytochrome P-450 rather than on microsomal electron transport.

Binding of benzo(a)pyrene metabolites to cellular DNA in perfused rat lungs

SummaryThe influence of pretreatment with monooxygenase inducers on total irreversible binding of metabolically activated [3H]-benzo(a)pyrene to cellular DNA and the formation of benzo(a)pyrene metabolite-deoxyribonucleoside adducts after cytochrome P-448 induction was studied in perfused rat lungs. Pretreatment with the cytochrome P-448 inducer β-naphthoflavone increasing binding by a factor of 23. In lungs of induced animals, 0.45 pmoles of benzo(a)pyrene equivalents were bound per mg DNA. Binding to RNA and to protein was also considerably induced by β-naphthoflavone. Phenobarbital treatment did not significantly increase binding to cellular macromolecules of rat lung. Analysis of hydrolyzed DNA of lungs from β-naphthoflavone-treated rats by Sephadex LH 20 chromatography revealed the formation of at least two nucleoside adducts with metabolically activated benzo(a)pyrene one of which is probably due to modification of the DNA with a benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and the other to modification of DNA with secondary metabolites of benzo(a)pyrene phenols.

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.

Effect of dietary antioxidants on benzo[a]pyrene metabolism in rat liver microsomes

Feeding of rats with 1% ethoxyquin (EQ) and butylated hydroxytoluene (BHT) but not butylated hydroxyanisole (BHA) increases the formation rate of benzo[a]pyrene (BP)-4,5-dihydrodiol from BP in hepatic microsomes. The production of other BP-dihydrodiols and of BP phenols is decreased after treatment with EQ, BHT and BHA. EQ and BHT are more effective than BHA in inducing epoxide hydrolase (EH) activity towards styrene oxide as the substrate.

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.

Veränderungen der Abbaugeschwindigkeit von Arzneimitteln und ihre Bedeutung für die Pharmakotherapie

SummaryIntensity and duration of drug action can be modified by altering the rate of drug metabolism. Alternative substrates of the hepatic drug metabolizing enzymes such as other drugs, steroids, and environmental chemicals, including insecticides and carcinogenic hydrocarbons, may either accelerate the biotransformation of a drug by enzyme induction or inhibit its breakdown by competing for the hydroxylation reaction. Xenobiotics known to stimulate or to inhibit drug metabolism in man are listed. Attention must be paid to induction and inhibition processes when drugs of a low therapeutic index are applied, e. g. coumarin anticoagulants, anti-convulsants, and oral antidiabetics. Administration of ethanol can also interfere with the rate of drug hydroxylation. Interactions between steroid inactivation and drug metabolism in the liver have frequently been demonstrated; their possible role in the treatment with contraceptive agents is discussed. Induction of the hepatic drug metabolizing enzymes has therapeutical implications for the detoxication of some physiological substances, e. g. bilirubin inicterus neonatorum or steroids in Cushings syndrome.Among the endogenous factors which have been shown to determine the rate of drug metabolism in animals and in man, genetic patterns, age, and endocrine influences from the adrenals, the gonades, and the thyroid as well as the metabolic situation of the liver in a hypoxic or diabetic state have to be mentioned. Liver diseases rarely impair the velocity of detoxication processes in the liver.ZusammenfassungWirksamkeit und Wirkungsdauer eines Arzneimittels können beeinflußt werden durch Veränderung seiner Abbaugeschwindigkeit. Alternative Substrate der arzneimittelabbauenden Enzymsysteme in den Lebermikrosomen, zu denen neben anderen Arzneimitteln auch Steroide und Umweltgifte wie Insecticide und carcinogene Kohlenwasserstoffe gehören, können den Stoffwechsel eines Arzneimittels einerseits durch Enzyminduktion beschleunigen, anderseits durch Konkurrenz um die Abbaureaktion hemmen. Fremdstoffe, deren induzierende bzw. hemmende Wirkung auf den Arzneimittelabbau beim Menschen nachgewiesen ist, werden aufgezählt. Besondere Beachtung erfordern Induktions- und Hemmvorgänge bei der Therapie mit Arzneimitteln geringer therapeutischer Breite, wie Anticoagulantien, Antikonvulsiva und oralen Antidiabetica. Alkoholzufuhr kann ebenfalls auf das Tempo des Arzneimittelstoffwechsels einwirken. Wechselwirkungen zwischen Steroidinaktivierung und Fremdstoffabbau in der Leber sind vielfach beschrieben; ihre mögliche Bedeutung bei der Anwendung von Ovulationshemmern wird diskutiert. Eine therapeutische Ausnutzung hat die Induktion der mikrosomalen Enzymsysteme bei der Entgiftung körpereigener Substanzen gefunden, z. B. bei der Prophylaxe des Neugeborenenikterus und beim Cushingsyndrom.Zu den endogenen Faktoren, deren Bedeutung für die Geschwindigkeit des Arzneimittelabbaus tierexperimentell und z. T. auch beim Menschen gezeigt wurde, gehören die genetische Ausstattung des Organismus, das Lebensalter, endokrine Einflüsse von seiten der Gonaden, der Nebenniere und der Schilddrüse und die Stoffwechsellage der Leber z. B. in der Hypoxie und beim Diabetes. Lebererkrankungen führen nur selten zur Beeinträchtigung der Entgiftungsfunktion der Leber.

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.