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J Magdalou

Publications and source records attributed to J Magdalou.

At least 55 records · Page 3Linked to original sources
PubMed1996

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.

Animals↗
PubMed1996

Inducibility of ethoxyresorufin deethylase and UDP-glucuronosyltransferase activities in two human hepatocarcinoma cell lines KYN-2 and Mz-Hep-1.

Two human hepatoma cell lines, KYN-2 and Mz-Hep-1 were characterized in terms of glucuronidation capacity and inducibility of cytochrome P4501A1/1A2 and several UDP-glucuronosyltransferases (UGTs). Cytochrome P4501A1/1A2 activity was measured using 7-ethoxyresorufin and that of UGTs with 16 different substrates. The effects of dimethyl sulfoxide (DMSO), 3-naphthoflavone, alpha-naphthoflavone, and rifampicin on these drug-metabolizing enzyme activities were studied. DMSO treatment increased in a dose-dependent manner the ethoxyresorufin O-deethylase (EROD) activity in KYN-2 cells, while an opposite effect was observed in Mz-Hep-1 cells. In KYN-2 cells, EROD was more responsive toward beta-naphthoflavone treatment in combination with DMSO. This activity was enhanced in Mz-Hep-1 cells more than 83 times by beta-naphthoflavone. The enhancement of EROD activity by DMSO and beta-naphthoflavone treatments of KYN-2 cells was abolished by alpha-naphthoflavone treatment. In Mz-Hep-1, only the inducing effect of beta-naphthoflavone was abolished by alpha-naphthoflavone treatment. Rifampicin treatment of KYN-2 cells reversed both the DMSO and beta-naphthoflavone effects on the EROD activity. Glucuronidation of steroids, bile acids, fatty acids and drugs was effective in KYN-2 and Mz-Hep-1 cells. Both 1-naphthol glucuronidation and the level of UGT1*6 protein detected by immunoblot and supporting this activity were lowered by DMSO treatment and increased by beta-naphthoflavone treatment in KYN-2 cells. In Mz-Hep-1 cells, DMSO and beta-naphthoflavone had no effect on 1-naphthol glucuronidation activity. DMSO, beta-naphthoflavone and rifampicin also affected the glucuronidation of various substrates supported by different UGT isoforms. These results indicate that KYN-2 and Mz-Hep-1 cells can be used as new in vitro models for the studies of drug metabolism and the regulation of the corresponding enzymes.

Antibiotics, Antitubercular↗
PubMed1996

Stereoselective irreversible binding of ketoprofen glucuronides to albumin. Characterization of the site and the mechanism.

We have previously shown that the acyl glucuronide of racemic ketoprofen can irreversibly bind in vitro to plasma proteins (Dubois, N., et ai., Drug Metab. Dispos. 21, 617-623, 1993), but the mechanism of the reaction has not been characterized. In the present study, the reactivity toward albumin of the glucuronide of both ketoprofen enantiomers was investigated. The extent of binding increased with the concentration of both protein and glucuronide. However, the two diastereoisomers showed different reactivities toward human serum albumin (HSA): the maximum yield of adducts with the glucuronide of the S-enantiomer was twice that obtained with the glucuronide of its antipode. The maximum extent of irreversible binding was at 4 hr for the R-ketoprofen conjugate, but was later for the S-form. Chemical modifications of albumin indicated that the glucuronide of the S-isomer reacted only with lysine residues, whereas the R-form linked covalently mainly with tyrosine residues and secondarily with lysine residues. A competition study using specific binding probes and fatty acids showed that the conjugates of S- and R-ketoprofen reacted with amino acids located in sites I and II of HSA, respectively. Taken together, these findings suggest that the irreversible binding of ketoprofen to albumin depends on the stereochemistry of the aglycon: the R-enantiomer binds to site II of the protein probably by a nucleophilic attack by tyrosine and/or lysine residues, whereas adduct formation via the conjugate of the S-enantiomer could occur at site I of HSA by the Schiff base mechanism. This irreversible binding at sites I and II may affect the major function of albumin (i.e. the transport of drugs and endogenous compounds).

Animals↗
PubMed1995

Characterization of a new class of inhibitors of the recombinant human liver UDP-glucuronosyltransferase, UGT1*6.

The inhibitory effect of a series of novel structurally related compounds on the human UDP-glucuronosyltransferase UGT1*6 stably expressed in a V79 cell line was investigated. The inhibitors contain a lipophilic N-acyl phenylaminoalcohol residue and a uridine moiety connected by a spacer varying for each compound. The effects of these compounds on the glucuronidation reaction measured with 4-methylumbelliferone as substrate were determined. The best inhibitor of the series, D-DPMSU, had an IC50 of 39 microM in the assay conditions. Low Ki values were found toward both UDP-glucuronic acid and 4-methylumbelliferone (17 and 21 microM, respectively). The inhibition was competitive toward both substrates. A similar strong and competitive inhibitory effect was observed with two other inhibitors, DHPASU and DHPASiU. Another compound, D-DPASiU, showed a pure competitive inhibition towards UDP-glucuronic acid, but a non-competitive inhibition towards the acceptor substrate. These data and the optimization of the structures of the inhibitors by molecular modeling suggest that D-DPMSU and DHPASiU compounds may be transition state analog inhibitors of the recombinant UGT1*6 enzyme.

Binding Sites↗
PubMed1995

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.

Animals↗
PubMed1995

Constitutive and inducible expression of drug metabolizing enzymes in cultured human keratinocytes.

Drug metabolizing enzymes, particularly those involved in the metabolism of carcinogenic chemicals, were characterized in cultured human keratinocytes. Using immunoblotting experiments, we analysed the expression of phase I enzymes, cytochrome P4501A1 (CYP1A1) and NADPH reductase, and phase II enzymes, phenol UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) isoform pi, in the presence of either classical inducers (i.e. 3-methylcholanthrene, dimethylbenz[a]anthracene, phenobarbital, and clofibrate) or all-trans retinoic acid (RA). This study has shown that the expression of CYP1A1 and UGT is concomitantly induced by 3-methylcholanthrene, dimethylbenz[a]anthracene, and RA, and that of NADPH reductase is only enhanced by phenobarbital and RA. In contrast, the expression of GST pi was not affected by the inducers. Using the reverse transcriptase-polymerase chain reaction, we have demonstrated that the effects of 3-methylcholanthrene, dimethylbenz[a]anthracene and RA on CYP1A1 expression correlate with an increase of CYP1A1 mRNA level. Our results indicate that, with the exception of clofibrate, xenobiotics and RA differentially modulate the expression of drug metabolizing enzymes.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1995

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)

Animals↗
PubMed1995

Stereoselective esterase activity of human serum albumin toward ketoprofen glucuronide.

Many carboxylic acid-containing drugs undergo conjugation with D-glucuronic acid in humans, leading to the formation of acyl glucuronides, which are excreted into urine. However, these metabolites can be hydrolyzed back to the parent aglycon; this reaction can be accelerated by human serum albumin (HSA). Although this phenomenon of interaction between the acyl glucuronide and HSA has been described for various drugs, the kinetics of the protein have not been characterized. The aim of this study was to investigate the HSA-mediated mechanism involved in the in vitro hydrolysis by albumin of the acyl glucuronides of (R)- and (S)-ketoprofen (a nonsteroidal anti-inflammatory drug), as model compounds. The conjugates of both ketoprofen enantiomers were incubated, separately or together, with increasing concentrations of albumin (14.5-145 microM) at pH 7.4 and 37 degrees. The reaction followed Michaelis-Menten kinetics and was stereoselective; the (R)-ketoprofen glucuronide was a better substrate than the S-conjugate. To identify the HSA domain involved in the hydrolysis reaction, specific probes of HSA binding sites were used as potential inhibitors. These probes, added at an equimolar probe/glucuronide ratio (145 microM), slightly decreased the hydrolysis (by up to 30%). They affected the reversible binding of (R)-ketoprofen glucuronide to HSA, as shown by CD studies. Because iodoacetic acid did not modify the single free cysteine residue on HSA, this amino acid residue cannot be the reactive one. In addition, the chemical modification of a single tyrosine residue (probably Tyr-411) on HSA by diisopropyl fluorophosphate significantly but weakly affected the hydrolysis of (R)-ketoprofen glucuronide, suggesting that this residue also is not involved in the catalysis. In contrast, the R-conjugate was not bound to modified albumin, as revealed in CD experiments. These results support the existence of distinct sites on HSA for reversible binding and hydrolysis of (R)-ketoprofen glucuronide.

Binding Sites↗
PubMed1995

Reduction and glucuronidation of naftazone by human and rat liver microsomes.

Reduction and glucuronidation of the vasoprotectant drug, naftazone, by human and rat liver microsomes and by recombinant UDP-glucuronosyltransferases (UGT) stably expressed in V79 cells were studied. The oxo group was first reduced in the presence of NADPH or NADH, and was subsequently readily glucuronidated on the phenolic moiety leading to a 1 beta-O-glucuronide, as revealed from MS and by proton and 13C-NMR. Glucuronide extracted from the urine of rats treated with the drug presented the same structure. In all enzyme systems tested, NADH was the most efficient electron donor, when compared with NADPH. The reaction was strongly inhibited by quercitrin, a specific inhibitor of carbonyl reductase. Attempts to isolate the reduced intermediate were unsuccessful because of its marked instability. In humans, a large interindividual variation for the formation of glucuronide was observed with microsomes of seven different liver samples (3.98 +/- 3.22 nmol/min.mg). In rat, glucuronidation of reduced naftazone was strongly induced (12-fold) by 3-methylcholanthrene and, to a lesser extent (2.6-fold) by phenobarbital, but was not affected by clofibrate. In addition, liver microsomes from Gunn rats, which present a genetic defect in bilirubin and phenol UGTs could not form glucuronide of reduced naftazone. The drug, after addition of NADH, was a substrate of the human liver recombinant UGT1*6 that presents a strict specificity toward planar phenolic substances, but not that of UGT2B4 and UGT2B1 expressed in V79 fibroblasts. The reducing step by the endogenous V79 cellular reductase was rate-limiting. In conclusion, the powerful inducing effect exerted by 3-methylcholanthrene, the lack of glucuronidation in the Gunn rat and the ability of UGT1*6 encoded by the UGT1 gene to glucuronidate reduced naftazone suggest that, in humans and in the rat, the compound is metabolized by a UGT isoform (UGT1*6 and the rat orthologous form) belonging to family 1, with a restricted specificity toward the drug.

Adult↗
PubMed1994

Stereoselective binding of the glucuronide of ketoprofen enantiomers to human serum albumin.

Since acyl glucuronides are known to undergo deconjugation, especially in the presence of human serum albumin (HSA), only a few reports have described their reversible binding to plasma proteins. The aim of this study was to investigate the reversible binding of R and S ketoprofen glucuronides to HSA by a rapid technique, such as ultraviolet circular dichroism. Binding of R ketoprofen glucuronide only induced an extrinsic Cotton effect at 340 nm. Scatchard plot analysis revealed that R ketoprofen and its glucuronide are bound to one site of albumin with an association constant of 28.1 x 10(4) and 6.1 x 10(4) M-1, respectively. Modification of one tyrosine residue by diisopropylfluorophosphate prevented the access of ligands to sites I and II of albumin, and also fully inhibited the binding of R ketoprofen and that of its conjugate. Displacement experiments with specific probes of albumin binding sites suggested that R ketoprofen and the glucuronide are bound to site II rather than site I. However, R ketoprofen was not displaced by its conjugate. S ketoprofen glucuronide is also bound to HSA, since it decreased the binding of the antipode conjugate. However, the binding of this metabolite to albumin did not induce an extrinsic Cotton effect large enough to determine the binding constants. D-Glucuronic acid did not bind to sites I or II of albumin. This moiety is likely responsible for the lower affinity of HSA for the R ketoprofen glucuronide when compared to that for R ketoprofen, due to the hydrophilicity and/or the bulkiness of this group.

Binding Sites↗
PubMed1994

Presence of a retinoid responsive element in the promoter region of the human cytochrome P4501A1 gene.

Cytochrome P4501A1 (CYP1A1) plays a key role in the metabolic activation of procarcinogenic compounds, leading to skin carcinogenesis. It is therefore important to determine whether its enzymatic activity is altered by topically administered drugs. We investigated, in cultured normal human keratinocytes (NHK), the effects of retinoic acid (RA) and synthetic analogs on the regulation of the CYP1A1 gene. Using transient transfections and gel shift assays, we demonstrated that the human CYP1A1 gene promoter was differentially regulated by retinoid receptors. We report, for the first time, that a RA responsive element 5'-CTTAGGTCACCACGGGGCA-3' (RARE1A1) is present within the promoter region of the CYP1A1 gene.

Base Sequence↗
PubMed1994

The chemical modification of human liver UDP-glucuronosyltransferase UGT1*6 reveals the involvement of a carboxyl group in catalysis.

The treatment of UDP-glucuronosyltransferase UGT1*6 stably expressed in V79 cells with three carboxyl-specific reagents, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K), resulted in a fast, dose-dependent decrease of the 4-methylumbelliferone glucuronidation. The inactivation reactions followed pseudo-first order kinetics. The pKa of the modified residue was close to 5.0. A partial protection against inactivation by Woodward's reagent was observed at pH 7.4 in the presence of UDP-glucuronic acid, UDP, and, to a lesser extent, in the presence of 4-methylumbelliferone. Dicyclohexylcarbodiimide significantly decreased the Vmax, without affecting the apparent Km towards UDP-glucuronic acid and 4-methylumbelliferone. The results support the involvement of a carboxyl group in the catalytic process.

Catalysis↗
PubMed1994

Photoaffinity labeling for evaluation of uridinyl analogs as specific inhibitors of rat liver microsomal UDP-glucuronosyltransferases.

The UDP-glucuronosyltransferases (UGT) involved in glucuronidation of endogenous and exogenous toxic compounds transfer the glucuronic acid residue from UDP-glucuronic acid (UDP-GlcUA), to various acceptor groups. A series of compounds that contain N-acyl phenylaminoalcohol derivatives linked to uridine or isopropylideneuridine were tested as UGT inhibitors. The potency of these inhibitors was determined by studying their effect on the photoaffinity labeling of rat liver microsomal UGTs by two photoaffinity probes, [beta-32P]5-azido-UDP-glucuronic acid (5N3UDP-GlcUA) and [beta-32P]5-azido-UDP-glucose (5N3UDP-Glc) and on the enzymatic formation of the two glucuronide conjugates (3-O- and carboxyl-specific) of lithocholic acid. All but one of the compounds tested proved to have an inhibitory effect on UGTs, both in the photoaffinity labeling system and in the enzymatic glucuronidation assay. In the photoaffinity labeling system, the inhibitors containing the isopropylidene moiety were less effective than their unprotected derivatives; however, the protected forms were, with one exception, more potent inhibitors of enzymatic activity. The photoaffinity labeling of UGTs with [beta-32P]5N3UDP-Glc was more susceptible to inhibition by all derivatives than that with [beta-32P]5N3UDP-GlcUA. The effect of one inhibitor, PP50B, on the two enzymatic activities involved in LA glucuronidation was extensively tested. A double-reciprocal plot suggested a competitive inhibition for UDP-GlcUA with an apparent Ki of 35 microM for LA 3-O-glucuronide formation and 94 microM for the carboxyl-linked glucuronide of the same substrate.

Affinity Labels↗
PubMed1994

High-performance liquid chromatographic enantioselective assay for the measurement of ketoprofen glucuronidation by liver microsomes.

A stereoselective high-performance liquid chromatographic (HPLC) method was developed to study the in vitro glucuronidation of ketoprofen enantiomers by liver microsomes. The HPLC system consisted of a Superspher 100 RP 18 end-capped column eluted with a mixture of acetonitrile and 10 mM tetrabutylammonium bromide in 1 mM potassium phosphate adjusted to pH 4.3 (30:70, v/v). Ultraviolet detection was performed at a wavelength of 254 nm. The capacity factors of S-ketoprofen glucuronide, R-ketoprofen glucuronide and R,S-ketoprofen were 12.8, 14.5 and 18.1, respectively. Sample pretreatment consisted of protein precipitation in microsomal incubation suspensions and further purification on a Sep Pak C18 cartridge before injection onto the HPLC system. Quantitation was performed with standard glucuronides biosynthetized with immobilized microsomes and purified by semi-preparative HPLC. The linearity of the method between 1.25 and 25.0 micrograms ml-1 (coefficient of correlation greater than 0.999), the repeatability (coefficient of variation = 1.2%; n = 5), and recovery (within 85%) were tested. The limit of detection was 10 ng for each glucuronide injected. The in vitro glucuronidation of R- and S-ketoprofen was measured in liver microsomes from man and from various animal species (dog, rat, rabbit). For both enantiomers, dog presented the highest specific activity. In contrast, the lowest activity was found in rabbit. On the other hand, the formation ratio of the S- and R-glucuronides of ketoprofen was close to 1 in man, rat and rabbit, but was 4.5 in dog, thus indicating that the reaction was stereoselective in this species.

Animals↗
PubMed1994

Expression of the human UDP-glucuronosyltransferase UGT1*6 in Escherichia coli. Influence of bacterial signal peptides on the production and localization of the recombinant protein.

The membrane-bound human liver UDP-glucuronosyltransferase UGT1*6 was expressed in Escherichia coli. Exchange of the natural signal peptide by the bacterial signal peptides of pclB or OmpT proteins considerably increased the level of expression and, as the natural signal peptide, targeted the protein to the membranes. The extent of maturation of SpelB-UGT1*6 precursor was about 30%. No processing of sOmpT-UGT1*6 occurred but the processing rate of this precursor could be significantly increased by mutagenesis of the first two amino acid residues of the mature sequence. These expression vectors allowed us to produce high levels of recombinant mature UGT1*6 required for further structural studies.

Amino Acid Sequence↗
PubMed1994

Chemical modification of human UDP-glucuronosyltransferase UGT1*6 by diethyl pyrocarbonate: possible involvement of a histidine residue in the catalytic process.

Chemical modification with diethyl pyrocarbonate (DEPC) of the recombinant human liver UDP-glucuronosyltransferase UGT1*6 in enriched membrane fractions from a V79 cell line resulted in a rapid inactivation of the glucuronidation reaction, measured with 4-methyl-umbelliferone as aglycone substrate, with a second-order rate constant of 3110 M-1.min-1 at pH 6.0 and 25 degrees C. The enzymatic activity was restored by hydroxylamine. Chemical modification with 0.2 mM DEPC for 60 s decreased the apparent Vmax 2.4-fold without significantly affecting the apparent Km toward 4-methylumbelliferone and UDP-glucuronic acid. Similarly, the binding of the photoactivatable cosubstrate analog [beta-32P]5-azido-UDP-glucuronic acid to the active site was not affected by the chemical modification. The enzyme was protected against this inactivation by 4-methylumbelliferone, suggesting that the modified residue was located in or near the aglycone binding site. In contrast, the cosubstrate UDP-glucuronic acid potentiated the irreversible inhibition, indicating a conformational change in the protein upon binding. The pH-dependence of the inactivation was in agreement with the modification of an amino acid residue with a pKa of 6.1. On the other hand, analysis of the variation of Vmax and Vmax/Km values of the glucuronidation reaction as a function of the pH revealed the presence of two essential residues with a pKa within the range 5.7-6.0. The data of the chemical modification of the recombinant enzyme together with that of the pH dependence of the activity strongly suggest the involvement of a histidine residue, highly reactive toward DEPC, which could be the base catalyst of the glucuronidation reaction supported by human UGT1*6.

Affinity Labels↗
PubMed1994

Expression and role of the human liver UDP-glucuronosyltransferase UGT1*6 analyzed by specific antibodies raised against a hybrid protein produced in Escherichia coli.

Characterization of human UDP-glucuronyltransferases (UGTs) has been limited by the unavailability of probes selective for each of several highly related isoforms. To better understand the role of this superfamily in the metabolism of drugs and xenobiotics, we describe a molecular/immunological strategy for discriminating the implication of each human isoenzyme in this process. Specific polyclonal antibodies were generated against the divergent amino-terminal domain of the UGT isoform UGT1*6 which is involved in the detoxification of nucleophilic compounds related to phenols and naphthols in human liver. The novel approach consists of the expression of a N-terminal UGT polypeptide fused to Staphylococcus aureus protein A in Escherichia coli and a single step purification of the fusion protein by immunoaffinity chromatography. Immunoblot and immunoinhibition analysis showed that the antibodies raised against the fusion protein selectively recognized both the denaturated and the native forms of UGT1*6, when expressed in V79 cell lines, but not three other recombinant UGT isoenzymes. In human liver microsomes, specific immunoinhibition analysis demonstrated that glucuronidation by UGT1*6 represented 20 to 50% of the total 1-naphthol UGT activity with a good correlation with the amount of protein selectively quantified on immunoblot. The specific expression of UGT1*6 was found to be significantly reduced in tumoral tissues but enhanced in cholestatic livers, when compared with healthy hepatic tissues. Interestingly, in human kidney microsomes, antibodies revealed a high level of UGT1*6 expression on immunoblot and inhibited 1-naphthol glucuronidation up to 55%, indicating that this isoform is also expressed in kidney and extensively contributes to phenol glucuronidation in this tissue.

Adult↗