Biomedical subjects

T Cresteil

Publications and source records attributed to T Cresteil.

At least 37 records · Page 2Linked to original sources

PubMed1995

Gene structure and promoter analysis of the rat constitutive CYP2C23 gene.

The CYP2C23 gene is expressed constitutively in the rat liver and kidney. It exhibits different profiles of expression in the two tissues, suggesting that several regulation processes could exist. In this paper, we report the structure of the 5'-flanking region of the CYP2C23 gene; 4.5 kbp were sequenced and analyzed. The CYP2C23 gene is present as a single copy into the rat genome and an unique transcription start site is used in both liver and kidney. Four DNase I hypersensitive sites have been mapped to the distal part of the hepatic promoter and three are detected in the kidney: only one site is present in the two tissues (L3/K1). In the proximal region, one site is specific for the kidney and one is detected in all the tissues tested. Footprint experiments allowed precise identification of the sequence of protected regions: HNF4 and CREB binding motifs are present in the distal liver-specific sites, motifs for AP-1, NF-1, and XRE-Bf are in the distal kidney site, and a Tf-LF1 binding site is localized in the L3/K1 protected site. In the proximal region, a sequence protected in all tissues contains a SP1/NF kappa B motif, whereas a sequence containing a HNF-4 binding motif is exclusively protected by kidney nuclear extracts. Altogether, the data clearly demonstrate that trans-acting factors involved in CYP2C23 gene expression differ in liver and kidney.

Animals↗

PubMed1994

Evidence for the transient expression of P-450 during the neonatal period in rat.

A cytochrome P-450 N (N for Neonatal) was resolved and partially purified from 15-day-old newborn rat livers. Antibody raised in rabbit against P-450 N detects two bands in liver microsomes, the major one comigrating with the partially purified P-450 N. This protein is not expressed in lung and kidney and exhibits an unusual ontogenic profile in the liver with maximal levels in 2-week-old animals. Expression of P-450 N is not modified in animals given beta-naphthoflavone or pregnenolone 16 alpha-carbonitrile, but is markedly increased by phenobarbital. Its amino-acid composition differs from that of other P-450s and it is not recognized by antibodies directed against CYP1A, CYP2A1, CYP2B, CYP2C11, CYP3A or epoxide hydrolase. It could thus be considered as an unique P-450 expressed transiently before puberty.

Amino Acid Sequence↗

PubMed1994

Taxol metabolism by human liver microsomes: identification of cytochrome P450 isozymes involved in its biotransformation.

The biotransformation of taxol by human liver was investigated in vitro with microsomes isolated from adult and developing human tissues. In vitro, no metabolism was detected with kidney microsomes, whereas two metabolites were generated by liver microsomes. The most prominent metabolite, termed M5, corresponded to an hydroxylation at the C6 position on the taxane ring, while the other metabolite, termed M4, corresponded to an hydroxylation at the para-position on the phenyl ring at the C3'-position of the C13 side chain. These two taxol derivatives have been shown to be the major metabolites recovered in bile from a patient infused with taxol. Several approaches have been used to identify the cytochrome P450 (CYP) isozymes involved in these reactions. No positive correlation was observed between the in vitro synthesis of these two metabolites, suggesting that two cytochrome P450 isozymes could be involved, although they could not be distinguished by their apparent affinities (Km approximately 15 microM). The formation of metabolite M4 was substantially reduced both by antibody directed against CYP3A and by the addition of CYP3A substrates such as orphenadrine, erythromycin, troleandomycin, and testosterone. Conversely, the formation of metabolite M5 remained unaffected by antibodies against CYP3A and by CYP3A substrates but was sensitive to diazepam inhibition, a preferential substrate of CYP2C. Correlation between CYP2C content or diazepam demethylation and the synthesis of metabolite M5 was highly positive. The formation of metabolite M4 developed during the early postnatal period. In contrast, the synthesis of metabolite M5 rose only after 3 months of age. These data clearly implicate CYP3A in the formation of metabolite M4 and CYP2C in the synthesis of metabolite M5. Microsomes from patients treated with barbiturates and benzodiazepines increased the formation of metabolite M4 to the level of metabolite M5, demonstrating that drug interactions could modify the human metabolism of taxol.

Age Factors↗

PubMed1994

A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identities.

A nomenclature based on comparisons of amino acid sequences is proposed for the members of the mammalian flavin-containing monooxygenase (FMO) gene family. This nomenclature is based on evidence of a single gene family composed of five genes. The percentage identities of the amino acid sequences of the five known forms of mammalian FMO are between 52 and 57% in rabbit and between 50 and 58% across species lines. The identities of all orthologs are greater than 82%. There is no evidence for multiple, highly related forms of the enzyme or for more than one mammalian FMO gene family. In the proposed system, the mammalian flavin-containing monooxygenase gene family is designated as "FMO" and the individual genes are distinguished by an Arabic numeral. The FMOs known as the "liver" and "lung" enzymes become FMO1 and FMO2, and the more recently described forms of the enzymes become FMO3, FMO4, and FMO5. Human FMO gene designations, FMO1 and FMO3, remain unchanged, but the gene designated FMO2 becomes FMO4. Following convention, the genes and cDNA designations will be italicized and the mRNA and protein designations will be nonitalicized. The purpose of the proposed nomenclature is to provide for the unambiguous identification of orthologous forms of mammalian FMOs, regardless of the species or tissue in question. The proposed classification considers only members of the mammalian flavin-containing monooxygenase gene family and has no bearing on the generally accepted definition of a multisubstrate flavin-containing monooxygenase.

Amino Acid Sequence↗

PubMed1994

Biotransformation of caffeine in human liver microsomes from foetuses, neonates, infants and adults.

1. Caffeine metabolism was studied in human liver microsomes from foetuses (n = 10), neonates (n = 10), infants (n = 9) and adults (n = 5). Caffeine and its metabolites, 1-3-7-trimethyluric acid, paraxanthine, theophylline and theobromine, were assayed by h.p.l.c. Methoxyresorufin-O-demethylase activity (MEROD) was determined and immunoquantifiable levels of CYP1A2 were measured. 2. The formation of the dimethylxanthines by N-3, N-7 or N-1-demethylation was significantly less in foetuses, neonates and infants than in adults, as shown previously in vivo. The formation of 1-3-7-trimethyluric acid (C-8-hydroxylation) was not significantly different between age groups. The production of total dimethylxanthines, paraxanthine and theophylline increased significantly with age within the neonate-infant group over at least the 0-300 day range (rs = 0.739, 0.667, 0.682, respectively). These data differ from those reported in vivo which suggested that N-3 and N-7-demethylations matured at about 120 days. The difference in maturational profiles of each metabolic pathway suggests that the reactions depend on different isoenzymes. The delay in the maturation of N-1 compared with N-3 and N-7-demethylation is in agreement with previous in vivo data. 3. In the neonate-infant group, only N-3-demethylation correlated with both MEROD activity (rs = 0.681; P < 0.05) and CYP1A2 microsomal concentration (rs = 0.454; P approximately 0.05), suggesting that, as in adults, this reaction depends on CYP1A2. 4. In the foetal samples, the production of total dimethylxanthines, paraxanthine and theobromine decreased significantly (rs = -0.879, -0.767, -0.708, respectively) with increasing gestational age.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Age- and tissue-dependent expression of CYP2C23 in the rat.

A cDNA was isolated from a library prepared with 15-day-old rat liver RNA and was shown to correspond to CYP2C23 with some minor alterations which modified the open reading frame. The expression of CYP2C23 was examined in rat liver, kidney, lung and testis and during ontogenesis. An appreciable amount of CYP2C23 RNA was observed in liver and kidney but the age-dependent expression was quite different between these two tissues. In liver, expression reached its maximal value in early neonates and remained quite stable, while the increase was progressive in kidney before declining after 3 weeks of age. In the liver, classical inducers of cytochrome P-450 decreased the CYP2C23 RNA content. Experimental data confirm the classification of CYP2C23 as a constitutive member of the 2C subfamily and clearly establish its age- and tissue-dependent expression in rat.

Aging↗

PubMed1993

The role of cytochrome P450 3A (CYP3A) isoform(s) in oxidative metabolism of testosterone and benzphetamine in human adult and fetal liver.

Testosterone metabolism was studied in human adult and fetal liver microsomes. In fetal livers 6 beta-hydroxylase (6 beta OH) activity (1-2% of adult activity) and 2 alpha-hydroxylase (2 alpha OH) activity (about 40% of adult activity) were present. Also some fetal livers produced two unknown metabolites. Androstenedione was formed in all fetal livers studied (10-20% of adult activity). Testosterone hydroxylations at 6 beta-, 2 beta-, 15 alpha- and 15 beta-positions were associated with CYP3A isoform(s) in adult liver, because they were strongly inhibited by midazolam, a known substrate for CYP3A4 and by anti-CYP3A4 antibody. Fetal liver activities were consistently inhibited less than the activities in adult livers. The formation of androstenedione was not affected by these inhibitors in fetal or adult liver microsomes. Benzphetamine N-demethylase activity in the fetal livers was about 40% of adult activity. Anti-CYP3A4 antibody had no effect on that activity in fetal or in adult liver microsomes, whereas a monoclonal antibody 1-68-11 (generated against rat CYP2C11) slightly inhibited benzphetamine N-demethylase activity in adult liver. This study indicates that human fetal and adult liver are dissimilar in their testosterone metabolism pattern. The formation of androstenedione from testosterone in fetal liver may have a physiological role. Testosterone hydroxylases are less inhibited by anti-CYP3A4 antibody, midazolam and progesterone in fetal than in adult liver.

Adult↗

PubMed1993

CYP2D6- and CYP3A-dependent metabolism of dextromethorphan in humans.

The metabolism of dextromethorphan, a drug used to probe genetically determined CYP2D6 activity has been investigated in vivo and in vitro. In vitro, kinetic parameters were determined in adult microsomes: Km for the O-demethylation was much lower than for N-demethylation (7 versus 650 microM) but Vmax was comparable. Fetal liver microsomes actively catalysed the N-demethylation of dextromethorphan, with kinetic parameters (Vmax and Km) quite similar in fetal and adult microsomal preparations while the O-demethylation did not exceed 5% of adult activity. In microsomes, the N-demethylation was inhibited by antibodies raised against CYP3A subfamily members although fetal microsomes were much less sensitive to immunoinhibition than adult microsomes. In vivo, urinary excretion of dextromethorphan and its three demethylated metabolites was examined in 155 adult volunteers and compared between extensive (n = 144, 92.9%) and poor (n = 11, 7.1%) metabolizers. The O-demethylation to dextrorphan is the rate-limiting step of metabolism. In 2D6 poor metabolizers, the N-demethylation to 3-methoxymorphinan is slightly higher than in 2D6 extensive metabolizers but does not compensate defective O-demethylation. The frequency distribution histograms of dextromethorphan/dextrorphan and 3-methoxymorphinan/3-hydroxymorphinan metabolic ratios appeared bimodally distributed, reflecting the participation of CYP2D6 in the O-demethylation reaction. They clearly differed from the random distribution of dextromethorphan/3-methoxymorphinan, and dextrorphan/3-hydroxymorphinan ratios among the population. These data clearly suggest that the N-demethylation of dextromethorphan is dependent on CYP3A and that both CYP2D6 and CYP3A are involved in the overall metabolism of dextromethorphan.

Adult↗

PubMed1992

Alteration of cytochrome P-450 2C11-dependent testosterone metabolism in Gunn rat liver.

Cytochrome P-450 2C11 (CYP2C11) is the main isozyme present in adult male rat liver and specifically hydroxylates testosterone in positions 2 alpha and 16 alpha. In Gunn rats, this isozyme is recognized by the anti-CYP2C11 antibody but its activity towards testosterone is dramatically decreased. Moreover, peptide mapping, after digestion of microsomal fractions by V8 protease and probing with anti-CYP2C11 antibody, exhibit a pattern which differs from that obtained with Wistar rats. Taken together, data suggest that the protein sequence of CYP2C11 from the Gunn rat differs from that of the Wistar rat.

Animals↗

PubMed1992

Impairment of mitochondrial 5-aminolevulinic acid synthase activity in Gunn rat liver.

Gunn rats are characterized by hereditary hyperbilirubinemia and a decrease, when compared to Wistar rats, in hepatic heme pool size which could result from an alteration of mitochondrial functions. Unconjugated bilirubin present in Gunn rat liver did not modify either the ultrastructural morphology or oxidative metabolism of the mitochondria as compared to those in Wistar rat liver. However, 5-aminolevulinic acid synthase activity is reduced by nearly 40% in Gunn rat liver mitochondria, thus explaining the reduced size of the hepatic heme pool.

5-Aminolevulinate Synthetase↗

PubMed1992

Long-term culture of adult rat hepatocyte spheroids.

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.

Albumins↗

PubMed1992

Cytochrome P450-dependent metabolism of dextromethorphan: fetal and adult studies.

Dextromethorphan undergoes O-demethylation to dextrorphan and N-demethylation to 3-methoxymorphinan. 3-Hydroxymorphinan, a didemethylated compound, is secondarily formed. The O-demethylation pathway to dextrorphan is polymorphic and under CYP2D6 genetic control. Adult and fetal studies were performed to characterize the cytochrome P450 families involved in dextromethorphan metabolism and their ontogeny. In adult volunteers in vivo and in vitro studies demonstrate that the O-demethylation pathway to dextrorphan is dependent on CYP2D6 and is predominant in extensive metabolizers and defective in poor metabolizers of the drug. The N-demethylation pathway to 3-methoxymorphinan is accessory and is dependent on the CYP3A subfamily. In human fetal microsomes, CYP2D6 protein and activity are not detectable until birth, while CYP2D6 RNA is present in significant amounts before birth. CYP3A activity is detectable in large amounts as early as the 17th week of gestation. Fetal and adult members of the CYP3A subfamily have close, although different, properties, as demonstrated by immunoinhibition studies.

Adult↗

PubMed1991

Expression of CYP2D6 in developing human liver.

The CYP2D6 protein is a polymorphic isoenzyme involved in the biotransformation of several drugs including the probe drug dextromethorphan. The rise in the protein concentration, immunochemically determined with a specific antibody, was shown to occur within the first week following birth, whatever the gestational age at birth. In fetuses, the concentration of hepatic CYP2D6 protein was very low or undetectable in 70% of samples tested. In the remaining 30%, its concentration was comparable to that of newborns aged 1-7 days. This early rise was associated with spontaneous abortion in 70% of positive samples, whereas in fetuses with an intermediate CYP2D6 protein concentration, 80% were from induced abortions. The rise in CYP2D6 protein was associated with the developmental onset of dextromethorphan O-demethylation, but not N-demethylation, even if activity was lower in fetal than in neonatal and in adult liver microsomes. Lastly, the CYP2D6 RNA is detectable earlier than the protein and exhibits a peak of hepatic accumulation in newborns, before declining in adulthood. A positive correlation between RNA accumulation and protein concentration can be demonstrated only in the adult. This suggest that regulation is primarily at the transcriptional level, but cannot rule out the participation of post-transcriptional events in the regulation process throughout ontogenesis.

Adult↗

PubMed1991

Control of cytochromes P450 expression in Gunn rat liver: implication of the intracellular heme pool.

The absence of changes in the overall hepatic cytochrome P450 content after administration of 3-methylcholanthrene (MC) to congenitally jaundiced Gunn rats is believed to be related to a limited heme availability in this strain of rat. The amount of available heme, estimated by tryptophan pyrrolase activity, shows a substantial decrease in control Gunn versus control Wistar rats. This reduction is moderately enhanced by MC treatment in Gunn rats but is abolished after phenobarbital administration. Heme oxygenase activity is diminished in Gunn rats and consequently is not responsible for the decrease in the hepatic heme availability. These data point out that the depletion of the intracellular heme can lead to a limitation in the synthesis of cytochrome P450 isoenzymes in the MC-induced Gunn rat.

Animals↗

PubMed1991

High levels of expression of the NAD(P)H:quinone oxidoreductase (NQO1) gene in tumor cells compared to normal cells of the same origin.

NAD(P)H:quinone oxidoreductase (NQO1) is a flavoprotein which catalyzes the two-electron reduction of quinones and azo-dyes and thus prevents the formation of free radicals and toxic oxygen metabolites that may be generated by the one-electron reductions catalyzed by cytochrome P450 reductase. Analysis of RNA indicated 20- to 50-fold higher levels of NQO1 gene expression in the liver tumors and in the tissue surrounding the tumors of patients with hepatocarcinoma than in normal individuals. An approximately 50-fold higher level of NQO1 mRNA was also observed in human hepatoblastoma (Hep-G2) cells than in normal liver. By deletion mutagenesis in the human NQO1 gene promoter and subsequent transfection into hepatic and nonhepatic cell lines, a 1.42 kb DNA segment has been identified to contain cis-acting elements responsible for high levels of expression of the NQO1 gene in tumor cells.

Base Composition↗

PubMed1991

Hormonal regulation of microsomal flavin-containing monooxygenase: tissue-dependent expression and substrate specificity.

The substrate- and tissue-dependent hormonal regulation of flavin-containing monooxygenase (EC 1.14.13.8) was studied in male and female rats. Hypophysectomy of males reduced liver microsomal N,N-dimethylaniline N-oxidation, thiobenzamide S-oxidation, and imipramine N-oxidation, although the reduction was not as marked with the latter substrate. Castration also reduced flavin-containing monooxygenase-dependent activities, but not to the same extent as hypophysectomy. Administration of growth hormone or testosterone to hypophysectomized males only partially restored basal activities. In female rats, hypophysectomy had no effect on N,N-dimethylaniline N-oxidation or thiobenzamide S-oxidation and actually stimulated imipramine N-oxidation (98%). These effects were demonstrated to be tissue- and sex-dependent. For example, hypophysectomy markedly (300%) enhanced imipramine N-oxidation in male kidney and significantly decreased the same activity in male and female lung. Correlations between levels of the enzyme determined by immunoquantitation (with antibody to the rat liver enzyme) and activities toward these three substrates, in male and female liver, lung, and kidney, also provide evidence for the existence of multiple forms of flavin-containing monooxygenase, which appear to be under different hormonal regulation.

Animals↗

PubMed1990

Evidence for the presence of distinct flavin-containing monooxygenases in human tissues.

Catalytic activities and substrate specificity of flavin-containing monooxygenase were examined in human tissues. During incubation with imipramine, human hepatic microsomes efficiently carried out cytochrome P450-dependent reactions but not the formation of N-oxide, while in kidney imipramine N-oxide was the only metabolite formed during in vitro incubation. The production of imipramine N-oxide was essentially due to flavin-containing monooxygenase as shown by thermal inactivation. In contrast, thiobenzamide and dimethylaniline were actively transformed by both human liver and kidney flavin-containing monooxygenase. Neither the modification of pH nor the solubilization of microsomal membranes increased imipramine N-oxidation in human liver. Kinetic analysis indicated a poor affinity (about 7 mM) of human liver microsomes for imipramine versus 0.3 mM in kidney. Immunological studies were undertaken to support enzymatic data. Antibodies raised against rat liver flavin-monooxygenase reacted strongly with human kidney microsomes but extremely weakly with liver microsomes. The relative amount of immunochemically determined protein correlated well with imipramine N-oxidation activity. A dose-dependent inhibition of imipramine N-oxidation by anti-flavin-monooxygenase antibodies was observed in human kidney, as well as in rat kidney and liver. Taken together, the results can be interpreted by the possible existence in human tissues of distinct flavin-containing monooxygenases exhibiting a partial overlapping substrate specificity. The protein involved in imipramine N-oxidation is missing from human liver but actively carries out the reaction in kidney, while another protein catalyzes the oxidation of thiobenzamide and dimethylaniline in both tissues.

Animals↗

PubMed1989

Phenobarbital-inducible gene expression in developing rat liver: relationship to hepatocyte function.

The expression of phenobarbital-, pregnenolone 16 alpha-carbonitrile- and polycyclic aromatic hydrocarbon-inducible cytochromes P-450 and of phenobarbital-inducible UDP-glucuronosyltransferase was examined in developing rat liver. RNAs coding for these proteins were present in fetal rat liver and their respective concentrations remained quite stable in non-induced animals. Inducers differently affected the concentration of RNAs: clofibrate had no action, whereas methylcholanthrene was highly active in fetal liver. Induction by phenobarbital gradually increased during ontogenesis, in parallel with the augmentation of the number of hepatocyte cells in the liver. Our contribution definitively demonstrates that the ability of phenobarbital to enhance P-450 and UDPGT RNAs is strictly restricted to hepatocytes and remains roughly unchanged throughout ontogenesis. In addition, phenobarbital was also able to potentiate the inducing capacity of methylcholanthrene (i.e., raising the TCDD-binding protein) exclusively in hepatocytes. This is the first direct evidence that the number of hepatocytes in the liver, rather than a biochemical maturation, controls the expression of phenobarbital-inducible genes. Pregnenolone 16 alpha-carbonitrile was also effective as inducer in fetal and neonatal rats and its maximal effect was observed in 5-d-old neonates, suggesting a regulation mechanism temporally different from that of phenobarbital.

Animals↗