Influence of vitamin A status on the regulation of uridine (5'-)diphosphate-glucuronosyltransferase (UGT) 1A1 and UGT1A6 expression by L-triiodothyronine.

The uridine (5'-)diphosphate-glucuronosyltransferases (UGT) are involved in the phase II of various xenobiotics and endogenous compounds. They are responsible for glucuronidation of many substrates, especially including bilirubin (UGT1A1) and phenolic compounds (UGT1A6). We previously showed that the expression of both isoforms is regulated at the transcriptional level by thyroid hormone in rat liver. In this present study, effects of vitamin A dietary intake (0, 1.72, 69 microg retinol acetate/g food) on the regulation of UGT1A1 and UGT1A6 activity and expression by 3,5,3' triiodo-l-thyronine (l-T3) were examined in the same organ. Activities were determined toward bilirubin and 4-nitrophenol. UGT mRNA were analysed by reverse transcription and amplification methods (reverse transcription-polymerase chain reaction) and quantified by capillary electrophoresis. In rats fed a vitamin A-balanced diet, a single injection of l-T3 (500 microg/kg body weight) increased UGT1A6 mRNA expression whereas this hormone decreased UGT1A1 mRNA expression. In addition we observed that the specific effect of l-T3 on UGT1A1 and UGT1A6 was reduced in animals receiving a vitamin A-enriched diet and disappeared in those fed a vitamin A-free diet. The modulations observed in mRNA expression are concomitant with those found for UGT activities. Our results demonstrate for the first time the existence of a strong interaction between vitamin A and thyroid hormone on the regulation of genes encoding cellular detoxification enzymes, in this case the UGT.

6-Mercaptopurine pharmacokinetics after use of azathioprine in renal transplant recipients with intermediate or high thiopurine methyl transferase activity phenotype.

Prevention of allograft transplant rejection by the immunosuppressive 6-thiopurine drug azathioprine is limited by haematological toxicity (leucopenia or agranulocytosis). This toxicity is particularly apparent in subjects with low thiopurine methyltransferase activity (TPMTase) phenotype (1% in the Caucasian population). The thiopurine derivative 6-mercaptopurine is the active metabolite of azathioprine, and it would be of interest to measure, after validation of plasma measurements, the mean values of the pharmacokinetic parameters in transplant patients with high or intermediate TPMTase phenotypes (85 and 14% of the Caucasian population, respectively). We measured erythrocyte TPMTase activity in 103 kidney transplant recipients of high or intermediate phenotype and calculated, after a test dose of azathioprine, the mean values of the pharmacokinetic parameters for 6-mercaptopurine. We also compared these values with the same parameters from one subject with low TPMTase activity phenotype. The mean observed area under the plasma concentration-time curve (AUC) was 190+/-140 ng mL(-1) h and the elimination rate constant (Kel) was 1.92+/-1. The pharmacokinetic parameters (AUC, Kel, t1/2el (the elimination half-life)) of 6-mercaptopurine in transplant patients are normally distributed and suitable for acceptance as a gold standard value for this population of Caucasian transplant patients. It seems useful to calculate these parameters, representative of the systemic exposure of individual patients to the drug, before prescribing these subjects azathioprine immunosuppressive treatment. In subjects with low TPMTase phenotype these pharmacokinetic measurements could also be an index of dose reduction.

Comparative quantification of two hepatic UDP-glucuronosyltransferase bilirubin isoforms mRNAs in various thyroid states in rat.

The study was designed to compare the effects of 3,5,3' triiodo-L-thyronine (L-T3) on the levels of hepatic mRNAs encoding two UDP-glucuronosyltransferase bilirubin isoforms (UGT1*1 and UGT1*0) in rats, by reverse transcription and quantitative polymerase chain reaction (RT-PCR). The administration of L-T3 decreased the UGT1*O mRNA by 2.2-fold and that of UGT1*1 by only 1.4-fold. In contrast, thyroidectomy increased the UGT1*O mRNA level by twofold but did not change that of the UGT1*1 isoform significantly. Interestingly, treatment with a known inducer of UGT bilirubin, ciprofibrate, induced the hepatic mRNA levels encoding for the UGT1*0 isoform by 3.5-fold and for the UGT1*1 isoform by only twofold. The results indicate for the first time that, although UGT1*1 mRNA is indeed a major transcript, its level is weakly affected by these compounds. In contrast, the minor UGT1*0 form is much more sensitive both to the action of this drug and to changes in thyroid status. The data support the notion that the various members of exon1 of the UGT1 locus have their own individual regulatory region.

Opposite regulation of bilirubin and 4-nitrophenol UDP-glucuronosyltransferase mRNA levels by 3,3',5 triiodo-L-thyronine in rat liver.

The effects of 3,3',5 triiodo-L-thyronine (L-T3) on the constitutive levels of hepatic mRNA encoding two UDP-glucuronosyltransferase (UGT) isoforms implicated in the glucuronidation of planar phenolic substrates (UGT1*06) and bilirubin (UGT1*0) were investigated in rat liver. The amount of UGT mRNA was quantitated by reverse transcription and amplification methods (RT-PCR). Treatment with L-T3 significantly increased UGT1*06 and decreased UGT1*0 mRNA levels by 41% and 54%, respectively. The opposite situation was observed in thyroidectomised animals. A good relationship observed between UGT activity toward 4-nitrophenol and bilirubin and mRNA levels emphasizes the key role played by the thyroid hormone L-T3 on UGT expression.

In vitro glucuronidation of peroxisomal proliferators: 2-ethylhexanoic acid enantiomers and their structural analogs.

In order to investigate the glucuronidation of 2-ethylhexanoic acid (2-EHA), a metabolite of the plasticizer di-(2-ethylhexyl) adipate, by liver microsomes of several mammalian species including man, a gas chromatography method for the quantification of the corresponding glucuronides was developed. The variation coefficients for intra- and interassay repeatability were less than 3 and 7%, respectively. The rat liver UDP-glucuronosyl-transferase (UGT) presented similar Km and Vmax toward the two enantiomers. The glucuronidation of the racemate 2-EHA or its enantiomers was strongly increased up to six times by treatment of the rats with phenobarbital and, to a lesser extent, by 3-methylcholanthrene. In contrast, the treatment of the rats clofibrate did not modify the activity. The induction was not stereoselective. The Gunn rats, which present a genetic defect in the bilirubin UGT isoforms, were able to glucuronidate the drug as well as the congenic strain. Moreover, the UGT-2B1 isoform, stably expressed in V79 cells, glucuronidated 2-EHA in an appreciable amount. Interspecies comparison indicated that the most active glucuronidation of 2-EHA occurred in the dog and the rat. The lowest activities were observed in the man and the rabbit. In all species considered, except rabbit and guinea pig which glucuronidated the R isomer faster, the R and S enantiomers were glucuronidated to a similar extent. The glucuronidation activity toward compounds chemically related to 2-EHA increased as a function of molecular weight, but was not affected by the position of the methyl or the ethyl moiety on the hydrocarbon chain. A correlation between the glucuronidation rate of 2-EHA and analogs and the activity of PCoA oxidase was observed.

Characterization of the in vitro glucuronidation of flurbiprofen enantiomers.

To investigate the glucuronidation of the R- and S-enantiomers of the nonsteroidal antiinflammatory drug, flurbiprofen, by liver microsomes of several mammals, including humans, a new and reliable HPLC method for the separation and quantification of the corresponding diastereoisomeric glucuronides has been developed. Interspecies comparison revealed that the glucuronidation of flurbiprofen was highly efficient with liver microsomes of humans, monkeys, rats, and guinea pigs (in decreasing ranking order). Gunn rats, which present a genetic defect in the bilirubin UDP-glucuronosyltransferase (UGT) isoforms, were still able to glucuronidate the drug. The R-enantiomer was glucuronidated faster than the S-form by liver microsomes of rats and humans. Although the KM of glucuronidation of R- and S-enantiomers by rat liver UGT were in same order of magnitude (apparent KM 0.52 and 0.57 mM, respectively), the apparent Vmax's were significantly different (9.34 and 5.48 nmol/min.mg of protein). Regardless of the enantiomer considered, the glucuronidation of flurbiprofen was strongly increased up to 5-fold by treatment of rats with phenobarbital and, at a lower extent, by 3-methylcholanthrene. In contrast, the treatment of rats with ciprofibrate markedly decreased the activity. Glucuronidation of R-flurbiprofen was more enhanced by phenobarbital than that of the S-antipode. Each flurbiprofen enantiomer could weakly inhibit the glucuronidation of its antipode in a noncompetitive way. The apparent Ki was 0.51 mM with R-flurbiprofen as a substrate, and 0.37 mM with S-enantiomer. On the other hand, the rat liver UGT2B1 isoform, stably expressed in V79 cells, could glucuronidate flurbiprofen in an appreciable amount.(ABSTRACT TRUNCATED AT 250 WORDS)

[Determination of serum or plasma alpha-tocopherol by high performance liquid chromatography: optimization of operative models]. / Dosage de l'alpha-tocophérol sérique ou plasmatique par chromatographie liquide haute performance: optimisation du mode opératoire.

A previous multicentric study set up by the Société française de biologie clinique has emphasized the usefulness of a standardized procedure for the determination by high performance liquid chromatography of alpha-tocopherol in serum or plasma. In our study, we have tested every step of the different published procedures: internal standard adduct, lipoprotein denaturation and vitamin extraction. Reproducibility of results was improved by the use of tocol as an internal standard when compared to retinol or alpha-tocopherol acetates. Lipoprotein denaturation was more efficient with ethanol addition than with methanol and when the ethanol/water ratio was > or = 0.7. Use of n-hexane or n-heptane gave the same recovery of alpha-tocopherol. When organic solvent/water ratio was > or = 1, n-hexane enabled to efficiently extract, in a one-step procedure, the alpha-tocopherol from both normo and hyperlipidemic sera. Performances of the selected procedure were: detection limit: 0.5 microM--linear range: 750 microM--within run coefficient of variation: 2.03%--day to day: 4.76%. Finally, this pluricentric study allows us to propose an optimised procedure for the determination of alpha-tocopherol in serum or plasma.

Effects of simvastatin, a lipoprotein-lowering drug, on the hepatic enzymes involved in drug metabolism in the Wistar rat.

1. Simvastatin, a competitive inhibitor of 3-hydroxy-3-methyl glutaryl CoA reductase, lowers the plasma cholesterol level and has been approved for treatment of hyperlipoproteinaemia. 2. Simvastatin has been studied for its effects on hepatic microsomal drug metabolism in rat. No induction of 7-ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD) and of UDP-glucuronosyltransferases were found, in vitro, after administration of 0.5, 1.5 and 10 mg/kg per day for 22 days. 3. Epoxide hydrolases (microsomal and cytosolic) were also unchanged after treatment with simvastatin. 4. No increase of the palmitoyl CoA oxidase activity or of mitochondrial glycerol phosphate dehydrogenase activity occurred. 5. Fatty acid distribution in rat liver microsomal phosphatidylcholines showed a significant decrease of C16:1 and a significant increase of C20:4 acids.

Cyclosporine determinations in heart and liver transplant recipients: comparison of HPLC and FPIA methods on whole blood and plasma.

One hundred and eighty five whole blood samples were analysed for cyclosporine levels by fluorescence polarization immunoassay (FPIA) and high performance liquid chromatography (HPLC). 123 came from 4 heart transplant recipients (mean age +/- SD: 47.50 +/- 20.56 years) and 62 from 4 liver transplant recipients (44.50 +/- 16.52 years). FPIA was done on plasma and whole blood in heart transplant recipients, on plasma in the liver recipients. HPLC was always done on whole blood. The results show a good correlation between FPIA on plasma (y) and HPLC (x) in liver recipients (n = 62, r = 0.935, y = 1.23x + 70 ng/ml), slightly worse between FPIA on plasma (y) and HPLC (x) in heart recipients (n = 64, r = 0.610, y = 0.78x + 189 ng/ml) and mediocre for FPIA on whole blood (y) and HPLC (x) in heart recipients (n = 123, r = 0.566, y = 1.35x + 594 ng/ml).

Comparative and simultaneous effects of simvastatin and ciprofibrate on plasma lipid parameters and upon hepatic drug metabolizing and peroxisome proliferation marker enzymes in the male Wistar rat.

The effects of an associated treatment with Ciprofibrate and Simvastatin upon plasma lipid parameters, liver Mixed Function Oxidases enzymes and peroxisomal markers have been studied in male Wistar rats. The association was efficient upon triglycerides, but not upon cholesterol. The inducive and proliferative effects commonly exerted by Ciprofibrate (5 mg/kg/day) were not significantly modified by the simultaneous treatment with Simvastatin (10 mg/kg/day). The increase of the seric alanine amino transferase activity was however much more pronounced after the associated treatment than after single administration of Ciprofibrate or Simvastatin.

Nutritional factors of microsomal enzymatic induction: influence of lipidic components of the diet.

We compared the ability of two different diets containing 6 per cent of maize oil and 6 per cent of fish oil to modify: firstly the enzyme induction by phenobarbital and secondly the phenobarbital hydroxylation by the liver either in vivo or during in vitro perfusions. The presence of fish oil in the diet increased the cyt P 450 content and the bilirubin glucuronosyl transferase activity. The two induction effects promoted by the association of the phenobarbital treatment and the eating of the fish oil were not additive and it was found that the phenobarbital induction effect was decreased by the fish oil consumption. Phenobarbital and p-hydroxyphenobarbital kinetics were different in the two groups of animals. Phenobarbital was more slowly eluted in the fish oil fed than in the maize oil fed rats while p-hydroxyphenobarbital was more slowly eluted by the fish oil-fed rat livers.

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.

Methods of theophylline assay and therapeutic monitoring of this drug.

The purpose of this article is to review various analytical methods of monitoring plasma theophylline. This article was investigated by the "Drug Commission" of SFBC (Société Française de Biologie Clinique). The primary objective is to provide the "know-how", particular for this analysis, which allows the choice between various analytical methods available: immunochemical or physiochemical ones. The techniques described are not necessarily the best, they are approved and tested methods which are the most frequently used in routine practice. The proposed immunochemical methods are: absorption spectroscopy methods: Enzyme ImmunoAssay (EIA), Enzyme Multiplied ImmunoAssay Technique (EMIT); Reflectance spectroscopy method: Apoenzyme Reactivation Immunoassay System (ARIS); Fluorometry spectroscopy method: Substrate Labeled FluoroImmunoAssay (SLFIA); Fluorometry spectroscopy on solid base; Polarization fluorescence spectroscopy ImmunoAssay (FPIA); Turbidimetric measurements: Particle Enhanced Turbidimetric Inhibition ImmunoAssay (PETINIA); Nephelometric measurement: Nephelometric Inhibition ImmunoAssay (NIIA). And the proposed physicochemical methods are: High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC). The second objective is a review of pharmacological properties of theophylline, necessary for a good understanding of therapeutic drug monitoring: intestinal resorption, distribution, metabolism and elimination, drug interactions, dose/response relationship, physiopathological variations and proposed "predictive" "theophylline test". The authors conclude that because of the multiplicity of methodologies used in theophylline therapeutic monitoring the choice of one of them is not easy. The best way to compare different techniques available would be the use of a "reference material" for theophylline monitoring and a quality control network between different clinical pharmacological laboratories.

[Influence of thyroid status on microsomal and peroxysomal enzyme induction by fibrates in rats]. / Influence des états thyroïdiens sur l'induction des enzymes microsomales et peroxysomales par les fibrates chez le rat.

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.

Inductive effects of fenofibrate and metabolism of phenobarbital.

The inductive effects of fenofibrate (FF) and phenobarbital (PB) were investigated in male Wistar rats. FF treatment produced an inductive effect on liver weight, cytochrome P450 content, and aniline hydroxylase (AH) and bilirubin UDP-glucuronosyltransferase (UDP-GT) activities in liver microsome fraction. PB and FF inductive effects were additive on liver weight but were not additive on P450 microsomal concentrations. On the contrary, FF administration decreased the inductive effect of PB on bilirubin UDP-GT activity. When FF and PB treatment were coupled, plasma and liver PB concentrations were not affected, whereas OHPB concentrations, especially in liver homogenate, were greatly decreased. Thus it can be concluded that the production of OHPB from PB was probably not accelerated, but the elimination of OHPB, the main metabolite of PB, was considerably enhanced. These results are to be compared with recent reports of structure-dependent induction of bilirubin glucuronidation by arylcarboxylic acids chemically related to clofibrate.

[Ingestion of soybean epoxide oil. Effects on monooxygenases, epoxide hydrolase and activities of UDP glucuronosyltransferases in hepatic microsomes of the rat]. / Ingestion d'huile de soja époxydée. Effet sur les monooxygénases, l'époxyde hydrolase et les activités UDP glucuronosyltransférases des microsomes hépatiques chez le 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.

[Effects of 3,5,3'-triiodo-L-thyronine on the enzymes responsible for the metabolism of xenobiotics in the rat after increase in the dietary supply of cholesterol]. / Effets de la 3,5,3'-triiodo-L-thyronine sur les enzymes responsables du métabolisme des xénobiotiques chez le Rat aprés augmentation de l'apport alimentaire en cholestérol.

A cholesterol rich diet fed to rats was found to increase the cytochrome P 450 content in hepatic microsomes. Furthermore, the variations of the same parameters promoted by 3,5,3'-triiodo-L-thyronine were strongly reduced in cholesterol supplemented rats. Cholesterol prevented the inducing effects of phenobarbital but did not oppose its decreasing effect on maximal fluorescence of ANS and PNA introduced into the microsome suspensions.

[Influence of 3,5,3'-triiodo-L-thyronine on the hepatic enzyme activities responsible for the metabolism of xenobiotics during the development of the chick embryo]. / Influence de la 3,5,3'-triiodo-L-thyronine sur les activités enzymatiques hépatiques responsables du métabolisme des xénobiotiques pendant le développement de l'embryon de Poulet.

The influence of thyroid hormones on microsomal drug metabolizing enzymes was studied in hypothyroid newborn rats and chick embryos. Administration of 3,5,3'-triiodo-L-thyronine strongly decreased the microsomal cytochrome P 450 content in hypothyroid new-born rats and thus could render the rat pup more susceptible to hepatotoxicity from drugs. The drug metabolizing system in 20 days old chick embryos was less sensitive to the effects of thyroid hormone, but administration of phenobarbital was accompanied by a strongly induction effect on microsomal enzyme activities.

Pharmacokinetics of bupivacaine after axillary brachial plexus block.

Severe cardiovascular complications have been associated with regional anesthesia using bupivacaine. Furthermore, in our practice, axillary brachial plexus block is commonly used for surgical emergencies of the hand in out-patients. The pharmacokinetics parameters: peak height (Cmax), peak time (tmax), half life of plasma elimination (t1/2), total apparent clearance (Cl), mean time of plasma retention (t) were studied in 20 male and female patients (mean age 35 and 32 years) receiving axillary local anesthesia by bupivacaine (BU) only (A group = 100 mg), B group BU = 200 mg, or BU + lidocaine (LI) (C group = BU 100 mg + LI 200 mg), plasmatic BU and LI were simultaneously measured by HPLC method. There were no significant differences in the pharmacokinetic data observed in groups A, B and C; total apparent clearances and half times of plasma elimination of BU after brachial plexus block are similar compared to those observed after i.v. administration. Absorption of BU seems to be total while the value of Cmax are very low compared to usual neurotoxic plasma levels, but near that of plasma concentrations observed in cardiac complications. All the tmax observed were under 2 h and the dose independence of the kinetic of BU is not admissible in this study. The association of LI induce no modification of the kinetic of BU. This work concludes that in this way of administration, the absorption of BU seems to be total. Pharmacokinetics of BU is dose dependent and the association of LI induces no significant modification of BU kinetics.