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Biomedical subjects

J Magdalou

Publications and source records attributed to J Magdalou.

At least 127 records · Page 7Linked to original sources
PubMed1984

Structural changes of liver microsomes in rat during neonatal life: influence on the glucuronidation rates of various substrates.

The Vmax of the membrane bound UDP-glucuronosyltransferase (UDP-GT) towards group 1 substrates (4-nitrophenol, 2-naphthol) was particularly higher in young rats than in adults. On the contrary, activity towards group 2 substrates such as borneol or testosterone was not detectable in fetus liver. The developmental pattern of UDP-GT was related to changes in lipid composition of microsomes, namely in the content in lysophosphatidylcholine which rose at birth. The phospholipid-cholesterol molar ratio also increased 2 fold from the 16th day of fetal life to the 4th day after birth. Measurement of the steady state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) as well as determination of the order parameter S and the rotation cone angle of the fluorescent probe strongly suggested an increase in membrane fluidity in rat liver microsomes during ontogenesis.

Animals↗
PubMed1984

Assessment of the Mulder and Van Doorn kinetic procedure and rapid centrifugal analysis of UDP-glucuronosyltransferase activities.

The optimal experimental conditions of the enzyme assay described by Mulder and Van Doorn (1975, Biochem J. 151, 131-140) for the measurement of UDP-glucuronosyltransferase activities were tested towards structurally different aglycones. This assessment of this assay revealed that addition of Triton X-100 as enzyme activator was necessary because of its apparent inhibitory effects on interfering reactions. Under these conditions, accordance of the data with results published in the literature was obtained. We present for the first time an UDP-glucuronosyltransferase assay adapted on a fast analyser centrifuge which allows a rapid and sensitive measurement of enzyme activity that is very useful for kinetic constant determination, without consuming a large volume of reagents.

Animals↗
PubMed1984

Kinetic properties of UDP-glucuronosyltransferase(S) in different membranes of rat liver cells.

Glucuronidation of 4-nitrophenol, borneol and morphine occurred in rough and smooth endoplasmic reticulum, Golgi apparatus and plasma membranes of rat liver cells. In all fractions, prior fixation of either substrate (UDP-glucuronic acid or the aglycone) enhanced the affinity for the second substrate. Whatever the membrane, glucuronidation of 4-nitrophenol was characterized by high Vmax and high affinity for UDP-glucuronic acid. On the other hand, glucuronidation of borneol exhibited a lower Vmax and a lower affinity for UDP-glucuronic acid. In the endoplasmic reticulum, conjugation of morphine had a low Vmax, but the enzyme had high affinities for both UDP-glucuronic acid and the aglycone.

Animals↗
PubMed1983

Induced hepatotoxicity in female rats by aflatoxin B1 and ethynylestradiol interaction.

Female Sprague-Dawley rats were treated with a single ip dose of aflatoxin B1 (AFB1) (3 or 6 mg/kg). Twenty-four hours later and weekly until killed, some of the rats treated with AFB1 were given ethynylestradiol (EE) by gavage at the dose of 13 mg/kg. One, three, six, and nine months following the beginning of the experiment, animals were killed. Light microscopy of liver and histochemical determinations of gamma-glutamyltransferase (GGT) as well as the measurement of hepatic drug-metabolizing enzyme activities were investigated. The results show that AFB1 induced only very weak changes in the levels of different constituents studied. Thus, the mycotoxin did not affect GGT activity and increased epoxide hydrolase activity by a maximum of 42%. In contrast, EE significantly and progressively decreased (20 to 50%) the activity of UDP-glucuronosyltransferase (UDPGT) as well as the concentration of cytochrome P-450 and microsomal proteins. However, the estrogen increased the activity of epoxide hydrolase up to 150% as well as the activity of the hepatic (400%) and plasma (175%) GGT. The results also indicate that AFB1 amplified the EE-induced increase in liver weight and enhanced the depressive effects of the estrogen on microsomal proteins, cytochrome P-450, and UDPGT. Foci of cellular alteration which consisted of clear, acidophilic and basophilic cell lesions were seen in the livers of treated rats examined by light microscopy. These lesions were more prominent in the livers of animals given combinations of AFB1 and EE; they were accompanied by a strong intensity of GGT staining in the periportal area and a marked increase of the enzyme activity in the plasma (324%). From the sixth month, the livers of some animals treated with the combinations of AFB1 and EE showed hyperplastic nodules. This study indicates that the interaction between chronic administration of EE and a single ip injection of AFB1 induces hepatic lesions considered as possible forerunners of liver cell carcinomas. It also shows that GGT is a potential marker of preneoplastic lesions and may be used, therefore, in epidemiologic surveys in humans exposed to liver carcinogens such as the aflatoxins.

Aflatoxin B1↗
PubMed1983

Comparative study of four hypolipidaemic agents on the activity of drug-metabolizing enzymes in rat liver microsomes.

In order to understand the secondary effects of hypolipidaemic agents in human therapy, the authors have studied the inductive properties of four of these drugs, clofibrate, F1379, fenofibrate and probucol, on hepatic drug metabolizing enzymes in the rat. Each hypolipidaemic molecule was administered once a day for five days at doses ranging from 100 to 450 mg/kg/day. All the drugs tested caused hepatomegaly, the effect being particularly marked in the case of F1379 and fenofibrate; on the other hand they decreased the microsomal protein content, especially after F1379 or probucol treatment. Cytochrome P-450 concentration was not greatly affected, with only a 40% increase by clofibrate (dose 200 mg/kg/day) and by F1379 at the lower dose. It is of interest that all the hypolipidaemic agents tested enhanced the activity of epoxide hydrolase with 4, 5 benzo(a)pyrene oxide as the substrate. Except for fenofibrate and probucol at the lower dose, they all strongly increased the activity. The greatest change was effected by F1379 which led to a three to eight-fold increase over the control values. We also measured UDP-glucuronosyltransferase activities using two substrates belonging to group I (4-nitrophenol) and group II (4-hydroxybiphenyl). It appears that the changes in enzyme activity found depended both on the type and the dose of the drug administered and on the chemical structure of the substrate. This study showed that hypolipidaemic drugs which are chemically related to clofibrate could greatly modify the activity of drug metabolizing enzymes and therefore alter the transformation of drugs administered concomitantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Conformational study of purified epoxide hydrolase from rat liver.

Hepatic epoxide hydrolase (EC 3.3.2.3) was purified from phenobarbital-treated rats by ion-exchange chromatography followed by hydrophobic chromatography. The enzyme had a specific activity of 300--400 nmol min-1 mg-1 protein with benzo[a]pyrene-4,5-oxide as the substrate. Circular dichroism (CD) spectra of the purified enzyme gave two negative bands, centered at 210 nm and 222 nm, respectively. The mean residue ellipticity at 222 nm was 12,9000 deg X cm(2) X dmol(-1), which indicated the presence of about 35% alpha-helical structures. Sodium dodecyl sulfate (SDS) greatly affected the shape of the CD spectra, which were gradually shifted to the blue. This suggested a decrease in the aggregation state of the protein. Electrostatic interactions were important in the organization of the enzyme structure since the conformation was stable between pH 7.4 and pH 10. At pH-values 5.0, 6.0 and 12.0, the CD bands underwent considerable changes in both amplitude and shape. Moreover there was a good correlation between the optimal pH range of the epoxide hydrolase activity and the organization state of the protein. After membrane reconstitution with liposomes, the conformation of the enzyme was not significantly modified by the presence of dimyristoyl L-alpha-phosphatidylcholine or other phospholipids. This constancy was obtained over a wide range of molar ratios of phospholipids to protein (0--500). However, phospholipids did increase the thermal stability of the enzyme. Fluorescence measurements of diphenylhexatriene (DPH) bound to dimyristoyl L-alpha-phosphatidylcholine indicated that addition of epoxide hydrolase modified the thermal transition of the lipid phase. On the other hand, electron paramagnetic resonance (EPR) signals of the nitroxide-labelled fatty acid, 2-(14-carboxy-tetradecyl)-2-ethyl-4,4-dimethyl-3,3,-oxazolidiny-oxyl, bound to the phospholipid, indicated that the presence of the protein decreased by about 53% the correlation time of the label, suggesting that its motion had increased. In conclusion, phospholipid-epoxide hydrolase interactions enhanced the fluidity of dimyristoyl L-alpha-phosphatidylcholine liposomes without changing the secondary structure of the enzyme. Electrostatic interactions also played an important role in the conformational stability of the protein.

Animals↗
PubMed1982

Effect of a dietary protein deficiency on the development of hepatic drug-metabolizing enzymes in young rats.

The effect of protein deprivation on the activities of hepatic drug-metabolizing enzymes was studied in young rats whose mothers had previously been on a protein-restricted diet during pregnancy. Dietary protein deficiency (5% lactic casein as the protein source) lowered the amount of cytochrome P-450 and the activities of epoxide hydrolase and UDP-glucuronosyltransferase (UDPGT) (l-borneol as the substrate) by about 45, 63, and 48%, for the first 8 weeks, respectively. Interestingly, UDPGT estimated with p-nitrophenol as the substrate was far less affected than that estimated from l-borneol glucuronidation. This finding provides further evidence of the heterogeneity of UDPGT. Restoration of a balanced diet for 15 days following protein deprivation quickly restored cytochrome P-450 and enzyme activities to control values. Our experiments showed that the development of drug-metabolizing enzymes was changed more by the diet in young rats than in older rats. This could affect the toxicity of drugs that are normally metabolized by these pathways.

Animals↗
PubMed1982

Phenobarbital induction of cytochrome P-450 and UDP-glucuronosyltransferase in rabbit liver plasma membranes.

The presence of cytochrome P-450 and UDP-glucuronosyltransferase was confirmed in rabbit liver plasma membranes. Their levels, expressed as amount or activity/mg protein, were 25 and 40% respectively of those found in hepatic microsomes. Both enzymes were induced by phenobarbital treatment of the animals: administration of the barbiturate for 4 days, produced a 150% increase in the cytochrome P-450 content and in the specific activity of UDP-glucuronosyltransferase. As compared with the microsomal enzymes, the plasma membrane enzymes were localized in a lipid environment characterized by high levels of cholesterol and sphingomyelin and a low level of phosphatidylcholine. Treatment with phenobarbital for 4 days also caused a significant increase of phosphatidylcholine and phosphatidylethanolamine content in plasma membranes.

Animals↗
PubMed1981

Effect of diphenylhydantoin and its main hydroxylated metabolite on the pharmacokinetics and the urinary and biliary excretion of phenobarbital and its p-hydroxy metabolite.

When rats which had been pretreated with a high dose of diphenylhydantoin (80 mg/kg) for 5 days were given a single intravenous dose of phenobarbital (30 mg/kg): (a) There was no increase in the rate at which phenobarbital (PB) disappeared from the plasma or the tissues of pretreated rats. (b) The percentages of phenobarbital and p-hydroxyphenobarbital (free and conjugated) excreted in the urine were similar in both treated and control animals. However, the percentage of conjugated p-hydroxyphenobarbital excreted, was almost twice that of the control group. (c) Pretreatment with diphenylhydantoin (DPH) markedly increased bile flow rates. Therefore these rats excreted more PB than their controls. The biliary excretion of hydroxylated metabolites of PB (free and conjugated), was similar to that found in urine. Hydroxylation was not increased although, there was a significant elevation in the percentage of conjugated metabolite excreted. In a study to establish whether the main metabolites of diphenylhydantoin interfered with the metabolism of phenobarbital the following results were obtained: (a) Intravenous administration of DPH together with PB caused a two-fold increase in the half-life of phenobarbital elimination. (b) Intravenous administration of PB, to bile duct cannulated rats which had been pretreated for 5 days with DPH, caused a significant reduction in the excretion of hydroxylated phenobarbital in comparison with their control group. However, all the excreted DPH was present in the conjugated form. (c) The DPH pretreated rats had significantly lower cytochrome P-450 and mono-oxygenase activities in their hepatic microsomes than the pretreated controls, and higher UDP-glucuronyltransferase activity with DPH itself as the substrate.

Animals↗
PubMed1981

Effect of aflatoxin B1 on hepatic drug-metabolizing enzymes in female rats. Interaction with a contraceptive agent.

1. The effect of a contraceptive on aflatoxin B1 toxicity has been studied in female rats treated for 15 consecutive days with repeated doses of aflatoxin (0.40 mg/kg/day), norethindrone (0.60 mg/kg/day) and ethynylestradiol (0.012 mg/kg/day). 2. Increases occurred in hepatic microsomal cytochrome P-450 content (31%), and in the activities of epoxide hydrase (77%), UDP-glucuronyltransferase (67%) and gamma-glutamyltransferase (78%). 3. Aflatoxin alone caused a 22% increase in GSH S-epoxide transferase activity, whereas the contraceptive given alone or combined with aflatoxin increased the hepatic reduced glutathione. 4. The effect of aflatoxin plus contraceptive was not additive. 5. The effects caused by aflatoxin and the contraceptive were similar, and the contraceptive (depending on its progestogen/estrogen ratio), may modify aflatoxin toxicity by increasing the drug-metabolizing enzyme activities and the concentration of hepatic glutathione.

Aflatoxin B1↗
PubMed1980

[Recent data on the metabolism of drugs by UDP-glucuronosyltransferases and sulfotransferases (author's transl)].

The authors review recent work on UDP-glucuronosyltransferases and sulfotransferases. UDP-glucuronosyltransferases are membrane enzymes which are strongly dependent on the lipid environment ; this is why they have only recently been isolated and purified. It seems at present, that at least two enzyme forms exist. The sulfotransferases are cytosol enzymes, more easily isolated. Their importance in the metabolism of drugs appears owing to an equilibrium with UDP-glucuronosyltransferases.

Animals↗
PubMed1979

Effects of membrane perturbants on UDP-glucuronosyltransferase activity in rat-liver microsomes. Circular dichroism studies.

Rat-liver microsomes were treated with two non-ionic detergents, Triton X-100 and Lubrol WX, with phospholipase A2, or with aqueous acetone solution. The activity of the membrane-bound UDP-glucoronosyltransferase (UDPGT, EC 2.4.1.17) was measured after the treatment with these perturbants. At the same time, modifications of the secondary structure of the microsomal proteins were followed and studied by circular dichroism (CD) spectroscopy. The detergents greatly activated UDPGT, maximally at a 1 mM concentration of either detergent. The maximally activating Triton X-100 treatment did not greatly change the ellipticity of the microsomes at 222 nm ((theta)222), whereas that with Lubrol WX affected the secondary structure of the membrane proteins more strongly. UDPGT activation also occurred in phospholipase A2-treated microsomes. Maximal activation was obtained after 1--5 min of incubation and was stable throughout the experiment. Phospholipase A2 at the ratio of microsomal protein to phospholipase 250 : 1 (w/w) slightly increased (theta)222 after 10 min of incubation and did not change it further even after 30 min of incubation. Treatment of liver microsomes with a 10 : 90 (v/v) aqueous acetone solution removed 90% of the total membrane phospholipids, particularly phosphatidylcholine and phosphatidylethanolamine. The UDPGT activity was decreased in lipid-depleted microsomes, and the enzyme was not reactivated when phosphatidylcholine-lysophosphatidylcholine liposomes were added at a low temperature. An even greater decrease was obtained when the lipid binding was carried out at 37 degree C. Lipid-depleted microsomes had a high (theta)222 associated with a red-shift of 2 nm, indicating partial aggregation of membrane proteins and an increase in the alpha-helical content of the protein after acetone extraction. However, this particular protein structure was partially reversible, since a binding of phospholipids to lipid-depleted microsomes gave a (theta)222 close to that found in control microsomes. The UDPGT activity was not dependent on the secondary structure of the membrane proteins.

Acetone↗