Search PubMed⌕ Search

Biomedical subjects

J Magdalou

Publications and source records attributed to J Magdalou.

At least 109 records · Page 6Linked to original sources
PubMed1987

Comparative study of clofibric acid and bilirubin glucuronidation in human liver microsomes.

Hepatic microsomal glucuronoconjugation of the hypolipidemic drug clofibric acid was characterized in human liver and compared to the acylglucuronide formation of an endogenous substrate, bilirubin. The affinity of UDP-glucuronosyltransferase for bilirubin was 15-fold higher than for clofibric acid; the Vmax for the transformation of the two substrates were similar. The analysis of the specific activity in 32 liver biopsies showed that glucuronidation of clofibric acid or bilirubin were comparable in man and in rat. However, UDP-glucuronosyltransferase activity towards clofibric acid exhibited a large interindividual variation in man. Sex or age did not influence the glucuronidation of bilirubin and clofibric acid. Among the drugs given to the patients only clofibrate was able to increase the bilirubin conjugation. No effect of alcohol or smoking on the conjugation of the two substrates was observed. The absence of correlation between UDP-glucuronosyltransferase activities towards clofibric acid and bilirubin together with the specific induction of bilirubin glucuronidation by clofibrate suggested that these arylcarboxylic substrates were conjugated by separate forms of UDP-glucuronosyltransferase in human.

Adult↗
PubMed1987

Differential time-course of induction of rat liver gamma-glutamyltransferase and drug-metabolizing enzymes in the endoplasmic reticulum, Golgi and plasma membranes after a single phenobarbital injection. Evaluation of protein variations by two-dimensional electrophoresis.

This study was conducted to follow as a function of time the activity of gamma-glutamyltransferase in the various membranes of rat liver cells after a single dose of phenobarbital (PB) (75 mg kg-1 body weight). Gamma-glutamyltransferase induction was maximal 24 h after PB treatment in both the rough endoplasmic reticulum and the plasma membranes. This pattern of induction differed from that of some drug metabolizing enzymes. While total cytochrome P-450 content was enhanced mainly in endoplasmic reticulum until 48 h after PB treatment, UDP-glucuronosyltransferase activity was not greatly altered by PB under the same conditions. The comparison of two-dimensional electrophoretic polypeptide profiles of each subcellular membrane isolated from control and phenobarbital-treated rats revealed important variations induced by PB. In plasma membranes, the heaviest subunit (apparent Mr = 60 x 10(3)) of hepatic gamma-glutamyltransferase was provisionally identified as a collection of polypeptide which differ only by their pI. The concentration of these polypeptides was smaller in the endoplasmic reticulum where they were of lower apparent molecular mass. This suggests that the gamma-glutamyltransferase precursor is already processed at the level of the endoplasmic reticulum but it is still not completely mature or glycosylated. Five days of continuous PB treatment induced by appearance of new gamma-glutamyltransferase isoforms in plasma membranes. We demonstrate that after a single injection of PB, gamma-glutamyltransferase activity increases simultaneously with some drug-metabolizing enzymes, such as total cytochrome P-450 but not with others, such as UDP-glucuronosyltransferases.

Animals↗
PubMed1987

Properties of human hepatic UDP-glucuronosyltransferases. Relationship to other inducible enzymes in patients with cholestasis.

Glucuronidation of 4-nitrophenol, nopol (a monoterpenoid alcohol) and bilirubin, which in the rat, are catalyzed by three different enzymes, has been examined in liver biopsies from patients with various liver diseases, in particular cholestasis. These different activities were not correlated, which strongly suggests that at least three independently regulated forms of UDP-glucuronosyltransferases were present in the microsomes. Non ionic detergents (Triton X100, Emulgen 911) and deoxycholate produced similar activation (more than 2-fold) of the glucuronidation of 4-nitrophenol. Amphipathic substances, such as CHAPS (3-[3-cholamidopropyl-dimethylammonio]-1-propane sulfonate), and lysophosphatidylcholines maximally increased this UDP-glucuronosyltransferase activity, the most potent being oleoyl lysophosphatidylcholine (4-fold increase). Discriminant analysis of the data revealed no correlation between the three different UDP-glucuronosyltransferase activities and the age or sex of the patients. A good correlation was found on multidimensional analysis between form 1 of the enzyme (4-nitrophenol glucuronidation) and, in decreasing order of magnitude, epoxide hydrolase (measured with benzo(a)pyrene-4,5-oxide as substrate), cytochrome P-450, 7-ethoxycoumarin deethylase, aspartate aminotransferase and gamma-glutamyltransferase (r = 0.89); and between Form 3 of the enzyme (bilirubin glucuronidation) and NADPH cytochrome c reductase, alkaline phosphatase, (r = 0.81). These relationships may reflect the differential variation in enzymatic activities in various hepato-biliary diseases.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1987

A new enzyme immunoassay of microsomal rat liver epoxide hydrolase.

Antiserum against purified rat liver microsomal epoxide hydrolase was produced in the rabbit. We developed an enzyme-linked immunosorbent assay which is reliable with regard to its analytical criteria. The concentration of epoxide hydrolase was measured in liver microsomes of control rats and animals treated with F 1379 (250 mg/kg/day) for 5, 7, 14, and 21 days. This hypolipidemic drug was able to induce strong epoxide hydrolase activity and enhance protein concentration. The gradual increase in epoxide hydrolase concentration paralleled the increase of epoxide hydrolase activity, with stabilization occurring after the 14th until the 21st day of treatment.

Animals↗
PubMed1987

Metabolism of tridiphane (2-(3,5-dichlorophenyl)-2(2,2,2-trichloroethyl)oxirane) by hepatic epoxide hydrolases and glutathione S-transferases in mouse.

The transformation of the herbicide tridiphane (Tandem, Dowco 356, 2-(3,5-dichlorophenyl)-2(2,2,2-trichloroethyl)oxirane by the epoxide-metabolizing enzymes, epoxide hydrolases (EH) and glutathione S-transferases (GST), was investigated in mouse liver microsomes and cytosol. The microsomal EH catalyzed the formation of tridiphane diol. The production of this metabolite was prevented by cyclohexene oxide at 1 mM, a known inhibitor of microsomal EH. The structure of the diol was verified by comparison of retention time or Rf of the compound with those of an authentic standard using gas-liquid chromatography or thin-layer chromatography techniques. The diol formed a diester with 1-butane boronic acid or an aldehyde with lead tetraacetate. Mass spectral analysis supported the structural assignment. After optimization of the assay conditions, kinetic constants for the hydration of tridiphane by the microsomal EH were determined (Km = 65 microM and Vmax = 0.9 nmol/min/mg protein). Dietary exposure of mice to the hypolipidemic drug clofibrate at a dose of 0.5% (w/w) for 2 weeks increased by 173% the metabolism of tridiphane to tridiphane diol by the microsomal fraction. No diol could be detected following incubation of tridiphane with the cytosolic EH, even after induction by clofibrate. Tridiphane was also a substrate for GST, but administration of clofibrate did not change the specific activity for the formation of the glutathione conjugate. The herbicide was a rather weak inhibitor of the microsomal EH and the cytosolic GST activities measured with cis-stilbene oxide and trans-stilbene oxide as substrates with I50's of 3.0 x 10(-5) and 1.8 x 10(-4)M, respectively. Tridiphane diol was a poor inhibitor of the enzymes studied, and the glutathione conjugate of tridiphane caused marked inhibition of only the GST activity (I50, 2.0 x 10(-5)M). By contrast the activity of cytosolic EH (trans-stilbene oxide) was relatively insensitive to the addition of tridiphane or of tridiphane metabolites.

Animals↗
PubMed1987

Differential induction profile of drug-metabolizing enzymes after treatment with hypolipidaemic agents.

Various hypolipidaemic agents differentially induced microsomal drug-metabolizing enzymes. Clofibrate, clofibric acid, fenofibric acid and dulofibrate, which are mainly hypotriglyceridaemic, increased the content in cytochromes P-450 (77-185% over control), and especially cytochrome P-452-dependent lauric acid 12-hydroxylation (5.6- to 8.4-fold increase). Bilirubin glucuronidation was 2.1- to 2.8-fold stimulated; epoxide hydrolase activity (benzo(a)pyrene-oxide) was only slightly increased by the drugs. By contrast, F1379, which lowers plasma cholesterol only, did not change cytochromes P-450 content and slightly affected the 12-hydroxylation of lauric acid. It dramatically enhanced the epoxide hydrolase activity (7.6-fold), and increased (200%) the glucuronidation of planar group I substrates (4-nitrophenol, 4-methylumbelliferone, 1-naphthol). These effects were accompanied by a highly positive staining of gamma-glutamyltransferase in the liver characterized by a great number of intensively coloured foci in the periportal and perilobular area of the tissue. Treatment of rats for three weeks with F1379 did not modify this typical profile in enzyme induction. Such continuous effect could reveal some biochemical changes of hepatocytes with important toxicological relevance. Compared to the parent compound, treatment of rats with two metabolites of F1379 led to a decrease in the induction potency on epoxide hydrolase and on the forms I of UDP-glucuronosyltransferase; by contrast, the content in cytochromes P-450 was increased.

Animals↗
PubMed1987

[Effects of the ingestion of oxidized fish oils on hepatic microsomal enzyme activities and membrane fluidity in rats. Influence of tocopherol overload].

Rats fed a dietary peroxidized fish oil showed an increase in cytochrome P-450 content and ethoxy-coumarin deethylase (ECDE) activity in liver microsomes. Administration of DL-alpha-tocopherol led to different effects according to the extent of the peroxidation in the fish oil. In rats fed a de-peroxidized oil, the inductive effect of phenobarbital on UDP-glucuronosyltransferase (UDGPT) activity was depressed by tocopherol. By the same time, induction of P-450 and ECDE remained unchanged, that of epoxide hydrase slightly increased. By contrast tocopherol strongly potentiated the inductive effect of phenobarbital toward UDPGT activity (group I substrates) in rats fed the peroxidized fish oil. The modification of the inductive effect of phenobarbital in combination with tocopherol on UDPGT activities was concomitant with an increase in seric transaminase activity and with a reverse effect as revealed from the study of the rate of fluorescent probes penetration in microsomes. The possible toxicity of the strong dose of tocopherol is discussed.

Animals↗
PubMed1986

Liquid chromatographic assay for the measurement of glucuronidation of arylcarboxylic acids using uridine diphospho-[U-14C] glucuronic acid.

A general method for the assay of UDP-glucuronosyltransferase activity towards arylcarboxylic acids (clofibric acid, 1- and 2-naphthylacetic acid) using UDP-[U-14C] glucuronic acid in liver microsomes is described. The 14C-labelled glucuronide was separated by high-performance liquid chromatography, identified by hydrolysis by beta-glucuronidase, characterized by laser desorption mass spectrometry and quantified by scintillation counting. The coefficient of variation of the enzyme activity for the inter-assay repeatability was below 4.5%. As little as 2.5 nmol of the arylcarboxylic acid glucuronides could be detected and precisely quantified. The method was applied to the determination of the apparent kinetic constants for glucuronidation of the acids. Clofibric acid was the best substrate for UDP-glucuronosyltransferase (Vmax/KM, the ratio of the maximum initial velocity and the Michaelis-Menten constant, is 12.3). The two isomers, 1- and 2-naphthylacetic acids, were transformed at a similar rate. However, they exhibited different enzymatic affinities, as the KM values were 1.0 mM and 5.6 mM for 1- and 2-naphthylacetic acid, respectively. This indicates that the spatial organization of the substrates played a critical role in this acyl glucuronoconjugation.

Animals↗
PubMed1986

Inhibition of bilirubin UDPglucuronosyltransferase activity by triphenylacetic acid and related compounds.

Bilirubin UDPglucuronosyltransferase of rat or human liver microsomes was inhibited, in vitro, by triphenylacetic acid and by structurally related arylcarboxylic acids. This inhibition appeared to be competitive towards bilirubin, and mixed-type towards UDPglucuronic acid. A decrease in the number of phenyl rings or the absence of the carboxyl group in the molecule gave structures which did not affect enzyme activity, showing that both the triphenyl moiety and the carboxyl group were necessary for the inhibition. On the other hand, successive additions of methylene groups in the aliphatic chain progressively increased inhibitory potency. Kappi,bilirubin for triphenylacetic acid was 96 microM compared with 5 microM for 7,7,7-triphenylheptanoic acid. The inhibition of bilirubin UDPglucuronosyltransferase was not due to displacement of bilirubin from albumin. On the basis of these results an attempt was made to delineate the molecular events leading to glucuronidation of bilirubin.

Animals↗
PubMed1986

Stereospecific induction of rat liver bilirubin UDPglucuronosyltransferase and lauric acid 12-hydroxylation by the isomers of 2-phenylpropionic acid.

The inductive effects of racemic 2-phenylpropionic acid and its isomers on rat liver bilirubin UDP-glucuronosyltransferase activity and lauric acid 12-hydroxylation (cytochrome P-452-dependent) were compared. The (S)-(+)-enantiomer and the racemic mixture gave the greatest induction of both enzyme activities, whereas (R)-(-)-2-phenylpropionic acid produced increases of only one-third of those of its antipode. The determination of the enantiomeric composition of the excreted 2-phenylpropionic acid after a single oral dose indicated that the (R)-(-)-enantiomer given as such or in the racemate was inverted to its antipode, which strongly suggests that (S)-(+)-2-phenylpropionic acid is responsible for the inductive effects observed. The demonstration of the same stereospecificity for the induction of bilirubin UDPglucuronosyltransferase and lauric acid 12-hydroxylation further indicates a close mechanistic link between these two processes.

Animals↗
PubMed1986

Kinetic constant determination of liver microsomal and purified UDP-glucuronosyltransferase after phenobarbital and 3-methylcholanthrene treatments in rats.

After induction by phenobarbital and 3-methylcholanthrene, UDP-glucuronosyltransferase involved mainly in the conjugation of planar substrates was purified. Compared to the microsomal enzyme, the purified protein exhibited less affinity towards the substrates, but the corresponding Vmaxs were increased. These results were attributed to a change in the lipid environment of the purified enzyme. The conjugation rate for 4-hydroxycoumarine was 15-45 times less than that measured for the 7-hydroxyisomer with the microsomal or the purified enzymes. Immunoprecipitation studies of the enzyme revealed that the two compounds were transformed by the same enzyme, or metabolized by two separate enzymes presenting the same antigenic site. The orientation of the hydroxyl group of planar aglycones in the active site is the determinant for the efficiency of catalysis.

Animals↗
PubMed1986

Modulation of UDPglucuronosyltransferase activity in rats by dietary lipids.

Male Wistar rats were fed for 40 d a purified diet whose lipid source (60 g/kg diet) was coconut, peanut, corn or fish (herring) oil. A low lipid (lipid-deficient) diet (corn oil, 2 g/kg diet) was also fed to some rats. There were no significant differences in final body weights of rats fed the coconut, peanut, and corn oil diets. Rats fed the fish oil diet gained less weight than those fed any other diet. However, liver weight, ratio of liver to body weight, and protein content were not affected by any of the diets. The plasma cholesterol concentration of rats fed fish oil was lower than that of the other groups of rats. This diet resulted in the highest cytochrome P-450 concentration and markedly enhanced epoxide hydrolase activity. No difference in the level in cytochrome P-450 was noted between the groups of rats fed the vegetable oils. Epoxide hydrolase activity was also significantly higher with the corn oil diet. Interestingly, only glucuronidation of group I substrates was stimulated by the fish or corn oil diets and lowered by the coconut oil diet. Liver microsomes of rats fed fish oil contained a high level of lipid peroxides; this diet greatly stimulated NADPH-dependent peroxidation. The differential stimulation of UDPglucuronosyltransferase activity towards group I substrates could be the results of a toxic action of the fish oil diet as suggested by the concomitant enhancements of epoxide hydrolase, transaminase activities and peroxide content.

Animals↗
PubMed1986

[Effects of ingestion of oxidized fish oils on membrane fluidity, and the activity of hepatic microsome enzymes in rats. Influence of vitamin A overload].

Oxidized fish oil (OFO) feeding is followed in the rat by the increase of vitamin A plasmatic level, and modifications of microsomal membranes (decrease of fluorophore ANS fixation). This inhibit the microsomal increase of cytochrome P 450, mixed function oxidase (MFO), any UDP-glucuronosyl transferases (group I) induced by phenobarbital treatment. On the other hand, vit A over load modify the microsomal structure of the opposite way (increase of ANS fixation). This effect is enlarged when OFO is added. In this case cyt P 450 is decreased. It seems that the magnification of vit. A effect is similar to increased microsomal membrane lability by oxidized fish oil addition.

Animals↗
PubMed1985

Structure-dependent induction of bilirubin glucuronidation and lauric acid 12-hydroxylation by arylcarboxylic acids chemically related to clofibrate.

The inductive potency of carboxylic acids, structurally related to clofibrate, on bilirubin UDPglucuronosyltransferase was investigated in the rat. For this purpose, structure-induction relationships were established using ten different arylcarboxylic or chlorophenoxycarboxylic acids. 4'-Chlorophenoxyacetic, -propionic and -isobutyric (clofibric) acids progressively increased hepatic glucuronidation of bilirubin (17%, 43%, 60% greater than controls, respectively) after a 5-day treatment in rat (100 mg/kg per day). 2-Phenylpropionic acid also enhanced bilirubin UDPglucuronosyltransferase activity (50%) in contrast to phenylacetic acid. The other compounds did not, or only slightly, affect this parameter. These results indicate that specific structural features are required for the induction property. Moreover, a good correlation (r = 0.962) was found between the extent of induction and the physiochemical descriptors which characterize the electronic state of the molecules, when analysed by multidimensional regression. Fluorescence polarization revealed that the compounds tested, especially clofibric acid, did not affect, in vivo or in vitro, the anisotropy of two different probes embedded in the microsomal membranes. Finally, since the interaction of the carboxylic acids with the membranes did not modify the latency state of bilirubin UDPglucuronosyltransferase, it was concluded that the increase in enzyme activity was due more to a real induction than to activation of bilirubin UDPglucuronosyltransferase. A close linkage was established between bilirubin UDPglucuronosyltransferase induction and that of cytochrome P-452, as shown by enhanced omega-oxidation of lauric acid. This led to the hypothesis that both processes could be under coordinate regulation and mediated by a molecular interaction depending on the physicochemical properties of the carboxylic acids.

Animals↗
PubMed1985

Conformational studies of NADPH cytochrome P-450 reductase by circular dichroism: interaction with phospholipids.

Ultraviolet circular dichroism spectrum of purified NADPH cytochrome P-450 reductase was characterized by two negative bands centered at 208 and 222 nm. The approximation of the alpha-helical content from the value of the mean residue ellipticity at 222 nm indicated 28% of alpha-helical structures. Heat inactivation of the enzyme was associated to a drastic change in the secondary structure of the protein. Membrane reconstitution experiments by inclusion of the enzyme into liposomes revealed that the conformation of NADPH cytochrome P-450 reductase was sensitive to its phospholipid environment. Egg lecithin as well as synthetic phosphatidylcholines, at the optimal phospholipid-enzyme molar ratio 200, was able to increase up to 37% the mean residue ellipticity at 222 nm. Addition of phosphatidylserine or phosphatidylethanolamine produced no effect. Non-ionic detergent such as Emulgen 913 weakly enhanced the mean residue ellipticity.

Animals↗
PubMed1985

[Effect of thyroid status on microsomal enzymes during liver regeneration in the rat].

The effects of thyroid status upon cyt. P 450 concentration and ethoxycoumarin deethylase, benzopyrene hydroxylase and UDP-glucuronosyltransferase activities in the liver microsome fraction were far more important in partially hepatectomized rats than in control animals. The partial hepatectomy simultaneously lowered the MFO enzymes activities in the hepatic microsome fraction and made them more sensitive to thyroid hormones effects.

7-Alkoxycoumarin O-Dealkylase↗