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E M Savenije-Chapel

Publications and source records attributed to E M Savenije-Chapel.

14 recordsLinked to original sources
PubMed1987

Inhibition of rat hepatic microsomal lipid peroxidation by mesna via glutathione.

Glutathione (GSH, 1 mmol/l) inhibits Fe2+/ascorbic acid induced liver microsomal lipid peroxidation. Oxidized GSH (GSSG, 1 mmol/l) did not affect rate or extent of lipid peroxidation. The inhibition by GSH seems specific since the sulfhydryl agent sodium 2-mercaptoethanesulfonate (mesna) gave a slight stimulation of lipid peroxidation. This stimulation is probably explained by a reduction by mesna of dehydroascorbic acid which is formed during the incubation, into ascorbic acid. Remarkably, mesna (1 mmol/l) added together with GSSG (1 mmol/l) produced the same inhibition as with 1 mmol/l GSH alone. This can be explained by direct reduction of GSSG to GSH by mesna. This is corroborated in experiments in which GSH is measured directly. Dimesna did not show an effect on lipid peroxidation. In the protective action of mesna against reactive substances its ability to reduce GSSG should be appreciated.

Animals↗
PubMed1984

Relationship between molecular structure and cytochrome P450-metabolic intermediate complex formation, studied with orphenadrine analogues.

Complexation of ferrous cytochrome P450 by metabolic intermediates formed during NADPH-catalyzed metabolism of compounds structurally related to orphenadrine was studied. This so-called metabolic intermediate complexation was determined in rat liver microsomes, obtained from phenobarbital-pretreated rats, at 455 nm using 33 microM of the orphenadrine derivatives. Using secondary amine derivatives with various N-alkyl substituents, a parabolic relationship between the logarithm of percentage of cytochrome P450 complexation and hydrophobic fragmental constant was observed. The derivative with a bulky tertiary butyl group, however, was devoid of metabolic intermediate-complexing activity. This indicates that steric factors besides lipid solubility may govern the complexing activity; also substitution at the phenyl group affects metabolic intermediate complex formation.

Animals↗
PubMed1984

Inhibition of mono-oxygenase and oxidase activity of rat-hepatic cytochrome P-450 by H2-receptor blockers.

Of four H2 blockers, cimetidine, tiotidine, oxmetidine and ranitidine, all except ranitidine showed ligand (type II) interactions with oxidized cytochrome P-450. High- and low-affinity binding sites were observed in hepatic microsomes of control, phenobarbital (PB)-treated and 3-methylcholanthrene (3-MC)-treated rats. All H2 blockers except for ranitidine (up to 400 microM) produced a concentration-dependent inhibitory effect of the metabolic intermediate (MI)-cytochrome P-450 complex formation which is displayed during metabolism of tofenacine in PB hepatic microsomes in vitro. At 400 microM, of all H2 blockers only oxmetidine was able to dissociate in vitro the isosafrole metabolite-cytochrome P-450 complex formed in vivo. Endogenous NADPH-dependent microsomal H2O2 production is inhibited in control, PB and 3-MC microsomes by the H2 blockers to various extents. In liver microsomes of phenobarbital-pretreated rats, substrate-dependent inhibition of H2O2 production correlates with inhibition of MI-cytochrome P-450 complex formation of tofenacine. Moreover, the magnitude of ligand (type II) binding of the H2 blockers correlates with inhibition of H2O2 formation. This indicates that prevention of oxygen activation by ligand binding decreases endogenous H2O2 production. Inhibition of both mono-oxygenase as well as oxidase activity of cytochrome P-450 may lead to adverse drug interactions. On the other hand formation of reactive or deleterious intermediates formed as a consequence of cytochrome P-450 activities can be prevented.

Animals↗
PubMed1983

Cytochrome P450 oxidase activity and its role in NADPH dependent lipid peroxidation.

A comparison is made between microsomal NADPH-dependent H2O2 production and malondialdehyde (MDA) formation in rat liver microsomes, obtained from phenobarbital pretreated rats. An increase in H2O2 formation was observed during NADPH-dependent disposition (10 min) of 100 microM diazepam (33%) and 2 mM hexobarbital (69%). In contrast orphenadrine (100 microM) and its mono-N-demethylated metabolite tofenacine (100 microM) decreased the H2O2 formation (35% and 55%, respectively). However, all these substrates were found to inhibit NADPH-dependent lipid peroxidation (60 min), estimated by measuring MDA formation, to various extents. These data strongly suggest that the oxidase activity of cytochrome P450 (H2O2 production) is not involved in a rate-limiting step in NADPH-dependent lipid peroxidation.

Animals↗
PubMed1983

Effect of multiple administration of orphenadrine or mono-N-desmethylorphenadrine on cytochrome P-450 catalyzed reactions in the rat.

Multiple administration (i.p.) of orphenadrine or its mono-N-demethylated metabolite, tofenacine (day 1, 20 mg/kg; day 2-5, 30 mg/kg) results in a considerable induction (50%) of the total cytochrome P-450 content. In addition, approximately 6% of the total amount of cytochrome P-450 was found to be blocked by a metabolic intermediate, formed from orphenadrine or tofenacine. Induction is apparent in enhancing the in vitro N-demethylation of aminopyrine and ethylmorphine and the p-hydroxylation of aniline. Pretreatment induced orphenadrine metabolism in vitro. The metabolism of tofenacine, however, was reduced. Probably this is due to a specific inhibition caused by the irreversible interaction of the metabolic intermediate with cytochrome P-450. In vivo, no induction of the aminopyrine metabolism (30 mg/kg, i.v.) is apparent, i.e., no change in the clearance was observed after pretreatment. This is probably due to the presence of relatively high, inhibitory concentrations of tofenacine (in the vicinity of cytochrome P-450). These results show that during chronic administration of orphenadrine or tofenacine, the in vivo disposition of concomitantly ingested compounds is determined by the influence of induction, high substrate and/or metabolite levels and complexation of cytochrome P-450. Moreover, based on these results an hypothesis is put forward in order to explain the phenomenon of product inhibition, which has been suggested to occur in man under chronic orphenadrine dosing conditions.

Aminopyrine↗
PubMed1983

Product inhibition in orphenadrine metabolism as a result of a stable cytochrome P-450-metabolic intermediate complex formed during the disposition of mono-N-desmethylorphenadrine (tofenacine) in the rat.

Product inhibition is thought to be involved in unexpected accumulation of orphenadrine, which occurs during chronic medication with this anti-Parkinson drug in man. In previous studies (Biochem. Pharmacol. 31, 2745-2753 (1982) we established the formation of reactive metabolic intermediates (MI) during metabolism of orphenadrine and its mono-N-demethylated metabolite tofenacine, which may block cytochrome P-450 (MI-complex). In this study we investigated the role of MI-complexation in product inhibition. Three different assays were used to establish the amount of cytochrome P-450 involved in MI-complexation, which was induced by tofenacine (30 mg/kg i.p.) in phenobarbital pretreated rats. If liver microsomes were prepared 3 hours after tofenacine injection, both spectral titration of oxidized cytochrome P-450, determination of loss of metyrapone binding sites at reduced cytochrome P-450 as well as ferricyanide oxidation of the MI-complex revealed 8-13% complexation of cytochrome P-450. Our data also suggest that MI-complexation is generated on phenobarbital induced cytochrome P-450 species. Phenobarbital induction was also shown to activate orphenadrine metabolism in vitro. Moreover, with a newly developed capillary GLC method, using nitrogen detection, we showed inhibition of orphenadrine- and tofenacine metabolism in vitro, by MI-complexation. This study therefore showed that MI-complexation may produce product inhibition.

Animals↗
PubMed1978

Sex differences in the kinetic constants of ethylmorphine demethylation and type I binding to hepatic microsomal cytochrome P-450 in mice. The influence of castration and testosterone.

1. In the CPB-SE mouse strain sex differences were observed in the Km and Vmax of ethylmorphine demethylation and in the deltaAmax of its type I binding to cytochrome P-450. In the CPB-V strain a small sex difference in the Vmax of the demethylation was found, whereas Ks and deltaAmax of type I binding differed considerably. 2. Testosterone pre-treatment of female CPB-SE mice abolished all sex differences, as did castration of males, except in Vmax, which was partially decreased. In the CPB-V strain testosterone pre-treatment of females abolished sex differences in type I binding, but had no effect on ethylmorphine demethylation. 3. Km values exceeded the corresponding Ks in all cases and sex differences in deltaAmax far exceeded those in Vmax. It is concluded that the Km is determined not only by the Ks of type I binding and the reduction rate of the type I complex between ethylmorphine and cytochrome P-450. The larger sex differences in deltaAmax as compared with Vmax may be attributable to type I binding of ethylmorphine to cytochrome P-450 subspecies not involved in its demethylation.

Animals↗
PubMed1977

Sex- and strain-dependent hepatic microsomal ethylmorphine N-demethylation in mice: the roles of type I binding and NADPH-cytochrome P-450 reductase.

The roles of type I binding and NADPH-cytochrome P-450 reductase in ethylmorphine demethylation were investigated in two strains of mice, using sex differences in these activities as a tool. In the CPB-SE strain, females metabolize ethylmorphine faster than males. Sex differences in cytochrome P-450 content and endogenous NADPH-cytochrome P-450 reductase activity were too small to account for this. On the other hand, the differences in the magnitudes of type I spectra and ethylmorphine-induced enhancement of cytochrome P-450 reduction were considerable larger than those in the rates of demethylation. All parameters, except endogenous cytochrome P-450 reduction, were modified in a similar way by testosterone pretreatment: in females they were depressed to the male level, whereas in males they remained unchanged. Castration had no effect in females and enhanced the activities in males. The CPB-V strain exhibited little or no sex differences in ethylmorphine demethylation, cytochrome P-450 content and endogenous cytochrome P-450 reduction. Testosterone pretreatment had little or no influence on these activities. Type I binding and reductase stimulation, however, showed sex differences, comparable to those observed in the CPB-SE strain, which were also abolished by testosterone. A relationship between reductase stimulation and type I binding was observed, which was, apparently, independent of sex or strain. It is concluded that androgen primarily influences the amount of cytochrome P-450-substrate complex formed, but that the reduction of this complex is not rate-limiting in the demethylation of ethylmorphine.

Animals↗
PubMedDate not supplied

Inhibition of diazepam metabolism in microsomal- and perfused liver preparations of the rat by desmethyldiazepam, N-methyloxazepam and oxazepam.

Hydroxylated metabolites of diazepam can be conjugated and are therefore generally thought not to affect the metabolism of diazepam. Liver microsomes, obtained from phenobarbital-pretreated rats, showed an inhibition of diazepam (10(-5) M) metabolism by desmethyldiazepam as well as by N-methyloxazepam or oxazepam (5 X 10(-5) M). In a single-pass perfusion of the rat liver an inhibition of diazepam disposition by exogenously administered desmethyldiazepam and by hydroxylated diazepam metabolites was also demonstrated. No oxazepam glucuronides were found after oxazepam infusion. However, infusion with N-methyloxazepam resulted in large amounts of oxazepam-glucuronides. The results indicate that administration of N-demethylated as well as hydroxylated metabolites may result in inhibition of the metabolism of their precursor. If hydroxylated metabolites are formed in situ they become more easily conjugated in comparison with administered hydroxylated metabolites and are therefore less effective as inhibitor.

Animals↗
PubMedDate not supplied

Dependence of hydrogen peroxide formation in rat liver microsomes on the molecular structure of cytochrome P-450 substrates: a study with barbiturates and beta-adrenoceptor antagonists.

In the present study, the molecular structure of xenobiotics has been successfully linked to their effect on the oxidase activity of cytochrome P-450, determined as microsomal hydrogen peroxide formation. A homologous series of 5-alkyl-5-ethyl barbiturates and a heterologous series of beta-adrenoceptor antagonists was used. The logarithm of the rate of microsomal hydrogen peroxide formation could be correlated with the logarithm of the apparent partition (n-octanol/buffer) coefficient for the barbiturate derivatives according to a parabolic function. The statistics of the correlation improved considerably by applying a bilinear model in order to fit the data. This probably indicates that both transport of the substrate to cytochrome P-450 and interaction with hydrophobic substrate binding sites of cytochrome P-450 are involved in the modulating effect of substrates on the oxidase function of cytochrome P-450. With the series of beta-adrenoceptor antagonists no clear-cut structure activity relationship with regard to the oxidase activity was apparent at first sight. However, when the inhibitory effect of the beta-antagonists on the 'cytochrome P-450 metabolic intermediate (MI) complex' formation that occurs during the microsomal biotransformation of 33 microM tofenacine was studied a relationship with the lipophilicity could be demonstrated. It is known that MI complex formation occurs with specific subforms of cytochrome P-450. By using this inhibitory activity of the beta-adrenoceptor antagonists, the interaction of the compounds becomes restricted to these specific subforms of cytochrome P-450. In both the oxidase activity as well as the MI complex formation phenobarbital induced cytochrome P-450 is involved.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗