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 considerab…

Effect of Monooxygenase Inducers on the Binding of Benzo-(A)Pyrene Metabolites to Cellular Macromolecules in Perfused Rat Lungs

The irreversible binding of metabolically activated [3H]-benzo(a)pyrene (BP) to cellular macromolecules of isolated perfused rat lungs was studied. Lungs from differently pretreated animals were perfused in situ in a recirculating system without ventilation. BP with a specific activity of 10 mCi/μmol was added to 50 ml perfusion medium containing 40% washed bovine erythrocytes to a final concentration of 1 μM. DNA, RNA and protein fractions were isolated and assayed for irreversibly bound radioactivity.

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.

Differential inhibition of biphenyl hydroxylation in perfused rat liver

A differential inhibition of biphenyl hydroxylation by alpha-naphthoflavone and metyrapone was observed in isolated perfused rat liver. alpha-Naphthoflavone inhibited 2- and 4-hydroxylation in livers from beta-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 beta-naphthoflavone-stimulated livers. Conjugation of 2-hydroxybiphenyl and 4-hydroxybiphenyl by isolated perfused livers was also studied. 4-Hydroxybiphenyl prefe…

Action of metyrapone on the redox state of free nicotinamide-adenine dinucleotide and on oxygen consumption of perfused rat livers and isolated mitochondria.

Metyrapone [2-methyl-1,2-bis-(3-pyridyl)-1-propanone] in a concentration of 5 mM increased the lactate/pyruvate ratio and theΒ-hydroxybutyrate/ acetoacetate ratio in the perfusion fluid of the isolated rat liver by a factor of 6 indicating a considerable shift in the ratio of free [NAD]/[NADH] in both the cytoplasmic and the mitochondrial compartment. Oxygen uptake of the isolated liver was decreased to about one half. The onset of the inhibitory effect on the respiration of the isolated organ was immediate. The inhibition lasted for at least 1 h.

Wirkungen von Metyrapon auf den Arzneimittelstoffwechsel und einige andere Funktionen der Leberzelle

ENHANCEMENT OF MICROSOMAL TYPE II SUBSTRATE OXIDATION BY HEMOGLOBIN

Ver�nderungen der Abbaugeschwindigkeit von Arzneimitteln und ihre Bedeutung f�r die Pharmakotherapie

Wirksamkeit und Wirkungsdauer eines Arzneimittels konnen beeinflust werden durch Veranderung seiner Abbaugeschwindigkeit. Alternative Substrate der arzneimittelabbauenden Enzymsysteme in den Lebermikrosomen, zu denen neben anderen Arzneimitteln auch Steroide und Umweltgifte wie Insecticide und carcinogene Kohlenwasserstoffe gehoren, konnen 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 aufgezahlt. Besondere Beachtung erfordern Induktions- und Hemmvorgange bei der Therapie mit Arzne…

Interrelationship between demethylation of p-nitroanisole and conjugation of p-nitrophenol in rat liver

The metabolism of p-nitroanisole (pNA) and p-nitrophenol (pNP) was studied in isolated rat livers perfused with a hemoglobin-free medium. The activity and viability of the surviving organ was tested by recording pH, “arterial” and “venous” oxygen tension as well as the disappearance of added pNP. pNA is converted to its primary metabolite pNP which, in turn, is excreted into the perfusion medium as conjugates. The coordination of pNA oxidation and the conjugation reactions of pNP were investigated. When 50 μM pNA is added as substrate 0.4±0.1 nmoles×ml−1×(g liver)−1 are excreted as pNP-glucuronide and 3.5±0.2 nmoles×ml−1×(g liver)−1 as the sulphate within 90 min. When pNP itself (50 μM) is …

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 demethylati…

Kinetic experiments on the binding of metyrapone to liver microsomes

Kinetic experiments on the inhibition of oxidative microsomal O- and N-demethylations by metyrapone (2-methyl-1, 2-bis(3-pyridyl)-l-propanone, Su 4885) were carried out using mouse liver microsomes as the enzyme source. The model substrates were p-nitroanisole and N-monomethyl-p-nitroaniline. It was shown that the inhibition is competitive. The K i for metyrapone is 0.42 × 10−4 M and for the reduced metabolite of metyrapone 1.15×10−4 M. Their spectral dissooiation constants as determined from difference spectra have almost the same values. From this it is concluded that the degree of inhibition is correlated to the amount of metyrapone bound to cytochrome P-450. Metyrapone does not seem to …

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 n…

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

The 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 beta-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 beta-naphthoflavone. Phenobarbital treatment did not significantly increase binding to cellular macromolecules of rat lung. Analysis of hydroly…

Experiments on the metyrapone reducing microsomal enzyme system.

The formation of reduced metyrapone [2-methyl-1.2-bis-(3-pyridyl)-1-propanol] from metyrapone [2-methyl-1.2-bis-(3-pyridyl)-1-propanone] in the liver has been studied. Reduced metyrapone appeared as the main metabolite of metyrapone in the isolated perfused rat liver. Experiments on the intracellular distribution of the metyrapone reducing activity revealed that metyrapone reductase is mainly localized in the microsomal fraction. In the 105,000 × g supernatant only a little activity was found.

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.

Similar level of metabolic activation of benzo(a)pyrene in perfused rat lung and liver and protection of lung by liver in a combined perfusion system

Abstract Irreversible binding of metabolically activated benzo(a)pyrene to DNA, RNA and protein proceeds by a different time course in perfused liver and lung of 5,6-benzoflavone-treated rats. Peak binding in liver is obtained after 15 min while binding in lung continuously increases over 120 min. Total irreversible binding per mg DNA or RNA is in the same order of magnitude in both organs. While binding in lung is lower at 15 min it exceeds binding in liver at 120 min. Binding per mg protein is higher in lung than in liver over the whole perfusion period. Introduction of a liver into the lung perfusion circuit decreases binding in lung. This protection effect is more pronounced when the li…