Coumarin 7-hydroxylase Activity Research Articles

Selective induction of coumarin 7-hydroxylase by pyrazole in D2 mice.

Pyrazole, was given to DBA/2N (D2), C57BL/6N (B6) and AKR/N mice to study its effects on hepatic drug metabolism. A decrease in the total amount of microsomal cytochrome P-450 as well as in the activities of ethylmorphine demethylase and benzo[a]pyrene hydroxylase was found. On the other hand ethoxycoumarin de-ethylase was increased 1.5-2.5-fold (depending on the strain of mouse) and coumarin 7-hydroxylase as much as sevenfold (but only in D2 mice) after pyrazole treatment. This increase was much higher than that caused by phenobarbital, the only well known inducer of coumarin 7-hydroxylase. By reconstituting the mono-oxygenase complex after purification of cytochrome P-450 we found a 40-fold increase in coumarin 7-hydroxylase and eightfold increase in ethoxycoumarin de-ethylase after pyrazole treatment. This was found only in D2 mice. An antibody previously developed against a cytochrome P-450 fraction from the the D2 strain with a high coumarin 7-hydroxylase activity inhibited the microsomal coumarin 7-hydroxylase almost 100% after pyrazole pretreatment of the animals. In the case of control or phenobarbital-treated mice the inhibition was somewhat weaker. With the reconstituted mono-oxygenase complex the inhibition of coumarin 7-hydroxylase was almost 100% both for control and pyrazole-treated D2 mice. The data indicate that pyrazole causes an induction of the microsomal monooxygenase complex different from that caused by phenobarbital or 3-methylcholanthrene and selective for coumarin 7-hydroxylation or 7-ethoxycoumarin de-ethylation. This induction was strong in D2, weak in B6 and absent in AKR/N mice.

Comparison of kidney, lung and liver coumarin 7-hydroxylases in phenobarbital pretreated DBA/2J and C57BL/6J mice

In this paper we describe catalytic and immunological properties of coumarin 7-hydroxylase, a cytochrome P-450 dependent enzyme activity, in the liver, kidney and lung of C57BL/67 and DBA/2J mice. Coumarin 7-hydroxylase activity was higher in D2 than in B6 mice in all three organs. For both strains of mice, liver had the highest enzyme activity when expressed per milligram of microsomal protein. However, when expressed per nmole cytochrome P-450 there was no difference in the enzyme activity between the tissues. Inhibition of microsomal coumarin 7-hydroxylase by antibodies previously developed in our laboratory against a cytochrome P-450 fraction from D2 and B6 mouse liver, associated with coumarin 7-hydroxylase, occurred as follows. In D2 mice both antibodies caused approximately 50% inhibition of the enzyme activity of all three organs. In B6 mice, however, the only organ where considerable inhibition took place was the liver, and only when antibody against B6 cytochrome P-450 was used. Ouchterlony immunodiffusion analysis revealed a 100% crossreactivity between the two strains of mice when similar organs were compared. The 100% crossreactivity was also found between the liver and lung in both strains of mice. However, only a 50% crossreactivity was found between kidney and liver or kidney and lung in B6 and between the kidney and lung in D2. The data demonstrate interorgan and interstrain differences in the immunological and catalytical properties of cytochrome(s) P-450 catalysing coumarin 7-hydroxylation.

Catalytic and immunological comparison of coumarin 7-hydroxylation in different species

1. 1. Coumarin 7-hydroxylation activities were determined in the liver microsomes of eight different species: pig, mouse (three strains), rat, hamster, guinea-pig, rabbit, rainbow trout and crayfish. 2. 2. A high coumarin 7-hydroxylase activity was found in the microsomes of D2 mouse, rabbit, hamster and pig, an intermediate activity in case of B6 and AKR mice, rat and guinea-pig and a very low activity in rainbow trout and crayfish. 3. 3. In Ouchterlony immunodiffusion analysis our antibody against coumarin 7-hydroxylase specific cytochrome P-450 from D2 mice was able to form a weak precipitin line only with rat and hamster microsomes in addition to the three strains of mice (D2, B6, AKR) where the band was very sharp. 4. 4. Strongest inhibition by the antibody (50% or more) of microsomal coumarin 7-hydroxylase was found in the case of the three strains of mice and rabbit. In the case of the other species the inhibition was very weak or did not exist.

Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver.

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.

Heterogeneous localization of two cytochrome P-450-dependent monooxygenase activities in dispersed cells of mouse lung

Cellular localization of two cytochrome P-450-dependent monooxygenase activities (7-ethoxycoumarin deethylase and coumarin hydroxylase) in the mouse lung was examined with the dispersed cells, which were fractionated on a Percoll isopycnic gradient and identified by electron microscopy. The 7-ethoxycoumarin deethylase activity was widely distributed in various types of lung cells. The coumarin hydroxylase activity, on the other hand, was almost exclusively localized in Clara cell-rich fractions (80% purity) in terms of both total and specific activities. Because coumarin hydroxylase is associated with cytochrome P-450, reportedly involved in the activation of various procarcinogens and necrotoxic substances, a high frequency of chemically induced cancer and necrosis in Clara cells may, at least in part, be explained by the exclusive localization of this enzyme activity in this type of cells.

Xenobiotic metabolism by alveolar type II cells isolated from rabbit lung

A procedure for the isolation of alveolar type II cells from rabbit lung was developed. Following pulmonary lavage to minimize macrophage contamination, viable cells (30% type II cells) were released by digestion with 0.1% Protease type I (Sigma) in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES)-buffered balanced salt solution containing 0.5 mM ethyleneglycolbis (amino-ethylether) tetra-acetate (EGTA) instilled via the trachea. Type II cells were enriched to 50–60 per cent purity by centrifugal elutriation. Density gradient centrifugation in metrizamide was used to increase the purity of the type II cells from the elutriator fraction to 80 per cent. Whole, freshly isolated alveolar type II cells metabolized 7-ethoxycoumarin at a rate of 30 pmoles umbelliferone formed per mg protein/min. However, only traces of coumarin hydroxylase activity were detected which could be accounted for by 1–2% Clara cell contamination in the type II cell fraction. NADPH-cytochrome c (cyt c) reductase activity in the sonicated type II cell fraction was 44 nmoles cyt c reduced per mg protein/min compared to 25 for lung homogenate. Benzo[ a]pyrene hydroxylase and N, N-dimethylaniline N-oxidase activities were also demonstrated in the type II cell fraction to be 13 pmoles 3-OH benzo[ a]pyrene · (mg protein) −1 · min −1 and 0.8 nmole DMA N-oxide · (mg protein) −1 · min −1 respectively. Microsomes prepared from the isolated type II cell fraction contained 74 pmoles cytochrome P-450/mg protein and 90 pmoles cytochrome b 5/mg protein.

Genetic regulation of coumarin hydroxylase activity in mice. Biochemical characterization of the enzyme from two inbred strains and their F1 hybrid.

Hydroxylation of coumarin to 7-hydroxycoumarin by liver microsomes from control or phenobarbital-pretreated mice is 5- to 10-fold higher in the DBA/2J strain compared to the AKR/J strain, while activities of nine other cytochrome P-450 mediated oxidations show only minor differences. Mixing experiments with whole liver homogenates and subcellular fractionations do not reveal the presence of enzyme activators or inhibitors or competing enzyme reactions in either strain. Comparisons of pH optima (pH 7.6), heat stability at 52 degrees C (6 to 8 min for 50% inactivation), and Km values (0.45 to 0.50 microM coumarin) for coumarin hydroxylase show no significant differences in the two strains of mice or their F1 hybrid. Similarly, only minor differences in inhibition of coumarin hydroxylase by carbon monoxide, SKF-525A, menadione, and several other inhibitors of microsomal mixed function oxidase reactions are observed in the two strains. In contrast to these data, aniline and metyrapone, two compounds which bind to the heme iron of cytochrome P-450 to form ferrihemochromes, show differential and opposite patterns of inhibition of enzyme activity in the DBA/2J and AKR/J mouse strains. This latter observation suggests that a structurally different cytochrome P-450 may hydroxylate coumarin in these two inbred mouse strains.

Genetic regulation of coumarin hydroxylase activity in mice. Evidence for single locus control on chromosome.

Genetic regulation of coumarin hydroxylase activity in mice. Evidence for single locus control on chromosome.

Genetic variation in coumarin hydroxylase activity in the mouse (Mus musculus).

Basal and phenobarbital-induced rates of hepatic metabolism of coumarin to 7-hydroxycoumarin are markedly higher in DBA/2J mice than they are in the AKR/J, C57BL/6J, and C3H/HeJ strains. Intermediate coumarin hydroxylase activity in F(1) hybrids of mating between DBA/2J and the other three strains indicates an additive mode of inheritance.

978. Species variations in coumarin hydroxylases: Creaven, P. J., Parke, D. V. & Williams, R. T. (1965). A spectrofluorimetric study of the 7-hydroxylation of coumarin by liver microsomes. Biochem. J.96, 390

The cynomolgus monkey is an animal species widely used to study drug metabolism because of its evolutionary closeness to humans. However, drug-metabolizing enzyme activities have not been compared in various parts of the liver and small intestine in cynomolgus monkeys. In this study, therefore, drug-metabolizing enzyme activities were analyzed in the liver (the five lobes) and small intestine (six sections from the duodenum to the distal ileum). 7-Ethoxyresorufin O-deethylation, coumarin 7-hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4′-hydroxylation, tolbutamide methylhydroxylation, S-mephenytoin 4′-hydroxylation, bufuralol 1′-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1′-hydroxylation, and testosterone 6β-, 16α-, 16β-, and 2α-hydroxylation were used as the probe reactions for this investigation. In liver, all probe reactions were detected and enzyme activity levels were similar in all lobes, whereas, in the small intestine, all enzyme activities were detected (except for coumarin 7-hydroxylase and testosterone 16α-hydroxylase activity), but from jejunum to ileum there was a decrease in the level of enzyme activity. This includes midazolam 1′-hydroxylation and testosterone 6β-hydroxylation, which are catalyzed by cynomolgus monkey cytochrome P450 (CYP) 3A4/5, orthologs of human CYP3A4/5, which are important drug-metabolizing enzymes. The data presented in this study are expected to facilitate the use of cynomolgus monkeys in drug metabolism studies.