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Biomedical subjects

J Magdalou

Publications and source records attributed to J Magdalou.

At least 91 records · Page 5Linked to original sources
PubMed1991

Expression of arylhydrocarbon hydroxylase, epoxide hydrolases, glutathione S-transferase and UDP-glucuronosyltransferases in H5-6 hepatoma cells.

1. The presence of arylhydrocarbon hydroxylase (cytochrome P-450 IA1 dependent), glutathione S-transferase, two distinct forms of epoxide hydrolases and UDP-glucuronosyltransferases was detected in H5-6 hepatoma cell homogenates using model substrates, selective inhibitors and specific antibodies. 2. The activity of arylhydrocarbon hydroxylase decreased strongly at the first days after plating and remained at a minimal value (1.5 pmol/min per mg) after 5 days of culture. 3. The hydratation of trans-stilbene oxide catalyzed by the soluble form of epoxide hydrolase was very low (11.0 pmol/min per mg), whereas the hepatoma cells contained appreciable amounts of the membrane-bound epoxide hydrolase and glutathione S-transferase measured with cis-stilbene oxide as substrate (maximal specific activity: 1.46 and 2.73 nmol/min per mg, respectively). 4. These cells also glucuronidated 1-naphthol efficiently (6 nmol/min per mg) and, at a lower extent, bilirubin (12 pmol/min per mg). 5. Addition of fenofibrate (70 microM) into the culture medium for 1-3 days failed to significantly stimulate the activity of cytosolic epoxide hydrolase. Only bilirubin glucuronidation increased 2-fold after 2 days of presence of the drug.

Animals↗
PubMed1991

Oral contraceptives stimulate the excretion of clofibric acid glucuronide in women and female rats.

1. Glucuronidation of clofibric acid, the pharmacologically active form of the hypolipidemic drug clofibrate was investigated in a human population, either in vitro with liver homogenates from biopsies, or after ingestion of the drug and determination of the urinary metabolite. No difference in the glucuronidation rate according to age of the patients was observed. Bilirubin but not clofibric acid glucuronidation was significantly higher in women (106% increase), when expressed per gram of tissue. 2. The excretion of clofibryl glucuronide in women who took oral contraceptives was significantly enhanced by 25%. 3. In female rats, treatment with the contraceptive agent norethindrone also stimulated by 48% the formation of clofibrylglucuronide in liver microsomes.

Adult↗
PubMed1990

Differential action of thyroid hormones and chemically related compounds on the activity of UDP-glucuronosyltransferases and cytochrome P-450 isozymes in rat liver.

The effect of thyroid hormones and chemically related compounds, on the activity of UDP-glucuronosyltransferases (EC 2.4.1.17) and cytochrome P-450-dependent monooxygenases in rat liver microsomes was investigated. The animals were thyroidectomized and treated with different doses of the drugs for 3 weeks. Opposite effects were observed depending on the isoenzyme of UDP-glucuronosyltransferase considered. While 3,3',5-triiodo-L-thyronine, 3,3',5-triiodothyroacetic acid, 3,3',5-triiodothyropropionic acid, isopropyldiiodothyronine and L- and D-thyroxine strongly increased 4-nitrophenol glucuronidation in a dose-dependent fashion, they decreased markedly bilirubin glucuronidation. However, the activity toward nopol, a monoterpenoid alcohol, was not significantly changed regardless of which compound or dose was used. Variation of UDP-glucuronosyltransferase observed with 4-nitrophenol and bilirubin was related to the thyromimetic effect of the drugs estimated from the increase in alpha-glycerophosphate dehydrogenase. Thyronine and 3,5-diiodo-L-tyrosine, which did not enhance this activity, also failed to affect glucuronidation. Variations in UDP-glucuronosyltransferase activity were more likely due to changes in protein expression rather than changes in enzyme latency, since lipid organization of the microsomal membrane, as estimated from the mean anisotropy of 1,6-diphenyl-1,3,5-hexatriene by fluorescence polarization was not significantly modified by the drug administration. Although some of the drugs could significantly decrease the triacylglycerol and cholesterol contents in plasma, all failed to affect lauric acid hydroxylation. The activities of catalase, palmitoyl-CoA dehydrogenase (CN- insensitive) and carnitine acetyltransferase in the fraction enriched in peroxisomes were also not significantly affected by treatment with the thyroid hormone LT3. In contrast, the activity of 7-ethoxycoumarine O-deethylase was increased by large doses of thyronine and by 3,3',5-triiodothyropropionic acid. The concentration of total cytochrome P-450 was decreased in a dose-dependent fashion by all the compounds used, except thyronine. Finally, significant correlations were observed between glucuronidation of bilirubin and 4-nitrophenol and the content in cytochrome P-450. This suggests a possible coordinate regulation of the two processes, which depends on the physicochemical characteristics of the thyroid hormones and related compounds.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1990

Phenobarbital inducible UDP-glucuronosyltransferase is responsible for glucuronidation of 3'-azido-3'-deoxythymidine: characterization of the enzyme in human and rat liver microsomes.

Glucuronidation by liver microsomes of 3'-azido-3'-deoxythymidine (AZT) was characterized in human and in various animal species. The glucuronide isolated by HPLC, was identified by mass spectrometry (fast atom bombardment, desorption in chemical ionization), and beta-glucuronidase hydrolysis. AZT glucuronidation reaction in liver microsomes of human and monkey proceeded similarly with an apparent Vmax of 0.98 nmol/min/mg protein and apparent Km of 13 mM. Oleoyl lysophosphatidylcholine activated more than twofold the formation of the glucuronide. Human kidney microsomes could also biosynthesize AZT glucuronide, although to a lower extent (six times less than the corresponding liver). Probenecid, which is administered to AIDS patients, decreased hepatic AZT glucuronidation in vitro (I50 = 1.5 mM), whereas paracetamol did not exert any effect at concentrations up to 21.5 mM. Morphine also inhibited the reaction (I50 = 2.7 mM). AZT glucuronidation presented the highest rate in human and in monkey (0.50 nmol/min/mg protein); pig and rat glucuronidated the drug two and three times less, respectively. In Gunn rat, the specific activity in liver microsomes was similar (0.18 nmol/min/mg protein) to that of the congenic normal strain; this suggests that an isozyme other than bilirubin UDP-glucuronosyltransferase catalyzed the reaction. In rats, AZT glucuronidation was stimulated fourfold by phenobarbital; 3-methylcholanthrene or clofibrate failed to increase this activity. This result was consistent with the bulkiness of the AZT molecule (thickness 6.7 A), which is a critical structural factor for glucuronidation of the drug by phenobarbital-induced isozymes. Altogether, the results strongly indicate that UDP-glucuronosyltransferase (phenobarbital inducible forms) is responsible for AZT glucuronidation.

Animals↗
PubMed1990

Human and murine cytosolic epoxide hydrolase: physical and structural properties.

1. Human and murine liver cytosolic epoxide hydrolase (CEH) had an apparent Mw of 59,000 by SDS-PAGE. 2. Peptide maps of CNBr, trypsin and Staphylococcus aureus V8 digests, as well as amino acid analysis, showed that human and murine CEH were similar. Uninduced and clofibrate induced murine CEH appeared qualitatively identical. 3. The CEHs shared antigenic determinants as determined by Western blotting. 4. Circular dichroism spectra indicate that human CEH had 39% alpha-helix. Uninduced and clofibrate induced murine CEH had 38 and 35% alpha-helix, respectively.

Amino Acids↗
PubMed1990

Reconstituted epidermis: a novel model for the study of drug metabolism in human epidermis.

The metabolic capacity of reconstituted epidermis from the outer root sheath cells of human hair follicles was determined. It was found that this epidermis possesses enzymes involved in both phase I (oxidation) and phase II (conjugation) reactions for drug biotransformation. The use of model substrates allowed the characterization of several isoenzymes. The homogenate fraction contained membrane-bound mixed-function oxydases (cytochrome P-450 dependent) involved in the O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzoxyresorufin, NADPH cytochrome c (P-450) reductase, testosterone 5 alpha-reductase, and UDP-glucuronosyltransferases, which conjugate 1-naphthol and bilirubin. One isoform of each glutathione S-transferase, steroid-, and arylsulfatases, acting on estrone- and 4-methylumbelliferone sulfates, was detected. Additionally, the activity of two distinct forms of epoxide hydrolases, which hydrate cis- and trans-stilbene oxides, could be measured. The presence of these drug metabolizing enzymes in the reconstituted epidermis indicates that it has a potential to serve as a model to study epidermal drug metabolism in vitro.

Arylsulfatases↗
PubMed1990

[Influence of thyroid status on microsomal and peroxysomal enzyme induction by fibrates in rats].

The influence of thyroid hormones upon the inductive effects on microsomal enzymes and the hepatic proliferation produced by different fibrates was studied in the male rat. A pharmacological dose of triiodo-L-thyronine significantly lowers the total cytochrome P450 content induced by some equi-effective doses of clofibrate, ciprofibrate, bezafibrate and fenofibrate. The ethoxycoumarin O-deethylase activity (ECOD) is concomitantly decreased. On the contrary, L-T3 accentuates the specific inductive effect of fibrates on bilirubin-UDPGT activity. Hypothyroid has little or no influence upon the effect of fibrates towards cytochrome P450 content and ECOD activity. On the other hand, this thyroid status significantly lowers the CN- palmitoyl coenzyme A dehydrogenase, which is a marker enzyme of the peroxisome proliferation caused by the different fibrates. These results indicate a possible modulation of thyroid hormones within the fibrates induction mechanism in the rat.

Acyl-CoA Dehydrogenase↗
PubMed1989

Evaluation of a versatile reversed-phase high-performance liquid chromatographic system using cethexonium bromide as ion-pairing reagent for the analysis of glucuronic acid conjugates.

An ion-pair high-performance liquid chromatographic technique has been developed to reduce the elution times of both glucuronic acid conjugates and their free aglycones. The chromatographic system combines the use of a reversed-phase column (LiChrospher CH-18; 5 microns) and a mobile phase of methanol (70-80%)-0.01 M phosphate buffer (pH 6.0) containing 2.5 mM cethexonium bromide as counter-ion at a flow-rate of 1 ml min-1. The hydrophobicity of this quaternary ammonium ion-pairing reagent and the high content of the organic modifier in the mobile phase provide close and short elution times for a wide structural variety of compounds (i.e. alcohols, phenols, steroids, carboxylic acids) and their conjugates with glucuronic acid (capacity factors lower than 7.5), without compromising the selectivity with respect to endogenous compounds of the microsomal incubation medium and urine. Advantages of cethexonium bromide over conventional tetrabutylammonium salts are clearly demonstrated, and the described system was applied to the simultaneous quantitation of clofibric acid and its acylglucuronide in human urine and validated for a pharmacogenetics purpose.

Animals↗
PubMed1989

Characterization of distinct forms of cytochromes P-450, epoxide metabolizing enzymes and UDP-glucuronosyltransferases in rat skin.

Study of drug metabolizing enzyme activity was undertaken in skin microsomal and cytosolic fractions of male and female rats. The presence of several isoforms was revealed from their activities towards selected substrates and from their cross immunoreactivity using antibodies raised against purified hepatic or renal cytochromes P-450, epoxide hydrolase and UDP-glucuronosyltransferases. Cytochrome P-450 content was precisely quantified by second derivative spectrophotometry, 23.1 and 16.5 pmol/mg protein in males and females, respectively. The monooxygenase activity associated to cytochromes P-450IIB1 and P-450IA1 was determined through O-dealkylation of ethoxy-; pentoxy- and benzoxyresorufin. The activity ranged between 4 and 2 nmol/min/mg protein for male and female rats, respectively. These results and Western blot analysis indicated that rat skin microsomes contain both monooxygenase systems associated with cytochromes P-450IIB1 and P-450IA1. By contrast lauric acid hydroxylation, supported by cytochrome P-450IVA1, was not detectable. Activities of epoxide metabolizing enzymes (microsomal and cytosolic epoxide hydrolases; glutathione S-transferase) were also characterized in skin. Microsomes catalysed the hydratation of benzo(a)pyrene-4,5-oxide and cis-stilbene oxide at the same extent, whatever the sex, although the specific activity was 10 times lower than in liver. The hydratation of trans-stilbene oxide by soluble epoxide hydrolase was four times lower than in the liver. Conjugation of cis-stilbene oxide with glutathione in skin and liver proceeded at essentially similar rates, as the specific activity of glutathione S-transferase in skin was only two times less than that measured in hepatic cytosol. Glucuronidation of 1-naphthol, bilirubin but not of testosterone could be followed in the microsomal fraction. Revelation by Western blot indicated that both the isoforms involved in conjugation of phenols and bilirubin were present in skin microsomes. By contrast, the isoform catalysing the conjugation of testosterone was apparently missing. When immunoblotting was carried out using specific antibodies raised against the renal isoforms, the same result was obtained. In addition, an intense staining corresponding to a 57 kD-protein was observed.

Animals↗
PubMed1989

Effect of hypolipidemic compounds on lauric acid hydroxylation and phase II enzymes.

Treatment of male Fischer 344 rats with various hypolipidemic drugs of different peroxisome proliferating potency (1-benzylimidazole, acetylsalicylic acid, clofibrate, tiadenol) led to an induction of liver lauric acid hydroxylase, whereas probucol, which is not a peroxisome proliferator, did not induce this enzyme. Activity of bilirubin UDP-glucuronosyltransferase was increased by all the compounds tested. The highest increase was observed after treatment with acetylsalicylic acid (2.3-fold). High correlation (r = 0.953) was observed between the activities of lauric acid hydroxylase and the corresponding activities of cytosolic epoxide hydrolase reported previously. The amount of microsomal epoxide hydrolase was not changed by any of the compounds. Whereas clofibrate and tiadenol decreased glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate, 1-benzylimidazole and probucol increased this activity. With 4-hydroxynonenal as a substrate qualitatively the same results were obtained with the exception that probucol did not affect the enzyme activity. When glutathione S-transferase activity was measured with cis-stilbene oxide as substrate only the more than five-fold increase after treatment with 1-benzylimidazole was significantly different from control values. Activity of dihydrodiol dehydrogenase was increased after treatment of rats with 1-benzylimidazole (1.5-fold), whereas application of tiadenol led to a decrease of enzyme activity. Feeding of male guinea pigs with clofibrate did not change the activity of peroxisomal beta-oxidation, cytosolic epoxide hydrolase or lauric acid hydroxylase. However, treatment with tiadenol caused an increase of these activities.

Animals↗
PubMed1989

Comparative induction of drug-metabolizing enzymes by hypolipidaemic compounds.

1. The inducing effect of hypolipidaemic compounds containing arylcarboxylic structure (fenofibrate, bezafibrate, ciprofibrate) on drug-metabolizing enzymes was compared with that of other chemically different molecules (probucol, 1-benzylimidazole) in rat liver. 2. Hepatomegaly was closely associated to the decrease in triglycerides and cholesterol contents in plasma. 3. Lauric acid hydroxylation and hydratation of trans-stilbene oxide by the cytosolic epoxide hydrolase were markedly increased by the arylcarboxylic acids (fenofibrate, bezafibrate and ciprofibrate). The determination of the concentration in microsomal epoxide hydrolase by immunoassay revealed no change in the protein amount within the membrane; only the specific activity was enhanced by bezafibrate and 1-benzylimidazole, suggesting an activation process. 4. Bilirubin glucuronidation was increased by the arylcarboxylic structures and by 1-benzylimidazole; by contrast probucol decreased this activity.

Animals↗
PubMed1988

Effect of 1-benzylimidazole on cytochromes P-450 induction and on the activities of epoxide hydrolases and UDP-glucuronosyltransferases in rat liver.

The influence of 1-benzylimidazole on the activities of hepatic monooxygenases cytochromes P-450 dependent, epoxide hydrolases and UDP-glucuronosyltransferases was investigated in male Wistar rats. Several doses (25, 75 and 100 mg/kg/day) were administered gastrically during 5 days in order to evaluate the dose-related induction. The treatment caused a dose-dependent hepatomegaly. 1-Benzylimidazole decreased the plasma level in triglycerides by 60-70%; by contrast the cholesterol content was not changed during the time course of the experiment. Lauric acid hydroxylase, benzphentamine N-demethylase, 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin O-deethylase activities were increased 3.5-, 4-, 13- and 46-fold, respectively with the highest dose. By immunoblotting, an enhancement in the protein bands corresponding to cytochromes P-450c and P-450b could be simultaneously observed, whatever the dose administered, thus suggesting an induction process. However, 1-benzylimidazole failed to bind with high affinity to the cytosolic Ah receptor. On the other hand, measurement of the activity of the microsomal epoxide hydrolase with benzo(a)pyrene-4,5-oxide as substrate and quantitation of the enzyme protein by immunoassay revealed that the increase in the activity after treatment with the compound was the result of enzyme activation only. By contrast, cytosolic epoxide hydrolase was not affected by 1-benzylimidazole. This compound also stimulated three distinct forms of UDP-glucuronosyltransferase. The activities towards 4-methylumbelliferone, 1-naphthol, morphine or a monoterpenoid alcohol, nopol, supported by two different isozymes were significantly increased only with the highest dose; meanwhile bilirubin glucuronidation was 2-fold enhanced, whatever the dose used. These observations emphasize the variety of the effects of 1-benzylimidazole on drug-metabolizing enzymes.

Animals↗
PubMed1988

1,2-Epoxycycloalkanes: substrates and inhibitors of microsomal and cytosolic epoxide hydrolases in mouse liver.

Six different 1,2-epoxycycloalkanes, whose rings were constituted of 5 to 12 carbon atoms, were tested as possible inhibitors of epoxide-metabolizing enzymes and substrates for the microsomal and cytosolic epoxide hydrolases (mEH, cEH) in mouse liver. The geometric configurations and the relative steric hindrances of these epoxides were estimated from their ease of hydrolysis in acidic conditions to the corresponding diols, their abilities to react with nitrobenzylpyridine, and the chemical shifts of the groups associated with the oxirane rings measured by proton and 13C-NMR. The cyclopentene, -hexene, -heptene, -octene and -decene oxides adopted mainly a cis-configuration. By contrast, cyclododecene oxide presented a trans-configuration. Steric hindrance increased with the size of the ring and was particularly strong when cyclooctene, -decene and -dodecene oxides were considered. With the exception of cyclohexene oxide, all the compounds were weak inhibitors of EH and glutathione S-transferase (GST) activities. Cyclohexene oxide exhibited a selective inhibition of the mEH with an I50 of 4.0.10(-6) M. As the size of the ring increased, inhibitory potency was gradually lost. The cEH and the GST activities were less sensitive to the inhibitory effects of these epoxides (I50, 1 mM or above). A marked difference between the substrate selectivities of mEH and cEH for these epoxides was observed. The mEH hydrated all of the cyclic epoxides, although some of them at a very low rate; the best substrate was the cycloheptene oxide (2.3 nmol/min/mg protein). On the other hand, cyclodecene oxide was a substrate of cEH, but no diol formation was detected when cyclopentene, -hexene and -dodecene oxides were incubated with cytosolic enzyme.

Animals↗
PubMed1988

[Ingestion of soybean epoxide oil. Effects on monooxygenases, epoxide hydrolase and activities of UDP glucuronosyltransferases in hepatic microsomes of the rat].

The effect of peroxidized soybean oil in the diet of male Wistar rats was studied on hepatic drug metabolizing enzymes and their phenobarbital induction and compared to that of natural soybean diet in the same conditions. No hepatomegaly or increase in serum transaminases occurred, however growth was inhibited after ingestion of peroxidized soybean oil. In addition, the protein biosynthesis of epoxide hydrase determined by immunochemistry was largely stimulated by this treatment; but the corresponding activity measured with benzo(a)pyrene 4-5 oxide as a substrate was increased in weaker proportions. This induction was limited to epoxide hydrolase only, since the enzymes of phase one were not affected and UDP glucuronosyltransferase activities toward group I substrates were randomly activated. The induction of epoxide hydrolase may affect only one or several isoforms of the membrane enzyme which are not necessarily specific to benzo(a)pyrene 4-5 oxide activity determination of the enzyme.

Animals↗