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Biomedical subjects

P Maurel

Publications and source records attributed to P Maurel.

At least 91 records · Page 5Linked to original sources
PubMed1992

Mitotic responsiveness of cultured adult human hepatocytes to epidermal growth factor, transforming growth factor alpha, and human serum.

The present study was undertaken to evaluate the ability of human hepatocytes to respond in culture to various mitotic agents including epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), or serum from patients with fulminant hepatitis. Human hepatocytes were maintained in culture on collagen-coated plates in a chemically and hormonally defined serum-free medium at low cell density. Twelve hours after plating, cultures were treated with increasing amounts of EGF (1-100 ng/mL), TGF-alpha (1-100 ng/mL), or human serum (1%-10%) for 0-96 hours. Proliferative response was assessed by determining against time the rate of DNA synthesis by [3H]thymidine incorporation in DNA, the labeling index, the expression of cyclin A, the amount of DNA, and the number of cells. The rate of DNA synthesis reached a maximum after 48 hours of treatment with 20 ng/mL EGF, 40 ng/mL TGF-alpha, or 5%-10% of human serum (fulminant hepatitis); the average increase with respect to untreated cells was 4.35 times with EGF, 5.4 times with TGF-alpha, and 4-6 times with serum from patients with fulminant hepatitis. The maximum expression of cyclin A coincided with the maximum of DNA synthesis. After 72 hours of treatment with EGF or human serum (fulminant hepatitis), the amount of DNA increased by 75%-100% (P less than 0.001) and the number of cells by 50% (P less than 0.001). These results show that adult human hepatocytes respond to mitogens, as expected from previous studies on animal hepatocytes, and provide experimental basis for future investigations in the field of human liver regeneration.

Adult↗
PubMed1992

Effect of corticosteroids on the expression of cytochromes P450 and on cyclosporin A oxidase activity in primary cultures of human hepatocytes.

Prednisone, prednisolone, and methylprednisolone are currently administered in association with cyclosporin A in the postoperative treatment of transplant patients. The aim of this work was to evaluate the effects of these corticosteroids on the expression of several forms of cytochromes P450 (P450), including P450 1A2, 2D6, 2E1, and 3A, and on cyclosporin A oxidase activity in human liver. For this purpose, human hepatocytes prepared from lobectomies were maintained in culture in a serum-free medium, in collagen-coated dishes, for 96-144 hr, in the absence or presence of 50-100 microM corticosteroids, rifampicin, or dexamethasone. To mimic more closely the current clinical protocol, hepatocyte cultures were also co-treated with corticosteroids and cyclosporin A or ketoconazole (a selective inhibitor of P450 3A). Cyclosporin A oxidase activity, intracellular retention of cyclosporin A oxidized metabolites within hepatocytes, accumulation of P450 proteins and corresponding messages, and de novo synthesis and half-lives of these P450 were measured in parallel in these cultures. Our results, obtained from seven different hepatocyte cultures, showed that 1) dexamethasone and prednisone, but not prednisolone or methylprednisolone, were inducers of P450 3A, at the level of protein and mRNA accumulation, as well as of cyclosporin A oxidase activity, known to be predominantly catalyzed by these P450; 2) although corticosteroids are known to be metabolized in human liver, notably by P450 3A, partial or total inhibition of this P450 by cyclosporin or ketoconazole, respectively, did not affect the inducing efficiency of these molecules; 3) corticosteroids did not affect the half-life of P450 3A or the accumulation of other forms of P450, including 1A2, 2D6, and 2E1; 4) chronic treatment of cells with cyclosporin did not affect P450 3A accumulation; 5) corticosteroids were all competitive inhibitors of cyclosporin A oxidase in human liver microsomes, with Ki values of 61 +/- 12, 125 +/- 25, 190 +/- 38, and 210 +/- 42 microM for dexamethasone, prednisolone, prednisone, and methylprednisolone, respectively; and 6) chronic treatment of cells with corticosteroids did not influence the excretion of oxidized metabolites of cyclosporin from the cells. These results support most of clinical reports dealing with mutual interactions between cyclosporin A and corticosteroids.

Adrenal Cortex Hormones↗
PubMed1991

Induction, regulation and messenger half-life of cytochromes P450 IA1, IA2 and IIIA6 in primary cultures of rabbit hepatocytes. CYP 1A1, 1A2 and 3A6 chromosome location in the rabbit and evidence that post-transcriptional control of gene IA2 does not involve mRNA stabilization.

A study on the regulation and induction of expression of cytochromes P450-IA1, IA2 and IIIA6 genes has been undertaken using primary cultures of adult rabbit hepatocytes grown in a serum-free chemically and hormonally defined medium. In 72-h-old cultures, 50 microM beta-naphthoflavone induced both IA1 and IA2 mRNA, the maximal level being reached after 4 h and 12 h, respectively. This was shown to result from an increase in the rate of transcription of gene IA1. In contrast, gene IA2 was constitutively transcribed in untreated cells, but mRNA only accumulated in the presence of beta-naphthoflavone which, however, did not affect the rate of transcription. Actinomycin D fully blocked induction of both IA1 and IA2 mRNA in response to their inducer. In untreated cells the presence of cycloheximide allowed a 'constitutive' expression of gene IA1, while in beta-naphthoflavone-treated cells, it produced a super-induction of IA1 but no modification of IA2 gene expression. Rifampicin (50 microM) strongly increased the IA1 mRNA level and rate of transcription only in cycloheximide-treated cells. Rifampicin and dexamethasone, two prototypical inducers of P450-IIIAs, induced both large and small IIIA6 mRNAs in a time-dependent fashion, the maximum level being reached after 24 h. This was related to a large increase in the rate of transcription of the gene. Cycloheximide significantly decreased the accumulation of both IIIA6 mRNAs in response to rifampicin, while actinomycin D fully blocked induction. The half-lives of IA1, IA2 and IIIA6 mRNAs were determined by two different methods, namely actinomycin D and [3H]uridine-chase experiments. In untreated cells, the half-lives for IA1, IA2 and IIIA6 mRNAs were 14 h, 16 h and 19 h, respectively when determined by the uridine chase and 18 h, 25 h and 22 h when determined by the actinomycin-D chase. These values were not modified significantly in cells treated with beta-naphthoflavone or rifampicin, indicating that neither of these inducers affected the stability of IA1 and IA2 or IIIA6 messages, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Developmental expression of rabbit cytochrome P450 CYP1A1, CYP1A2 and CYP3A6 genes. Effect of weaning and rifampicin.

Developmental expression of CYP1A1, CYP1A2 and CYP3A6 in the rabbit have been studied. Cytochromes P450IA1, P450IA2 and P450IIIA6 exhibited comparable patterns of developmental expression. Present at low level (less than 0.05 nmol/mg) in the new born animal up to week 3, these proteins sharply accumulated between weeks 3 and 4 to reach a maximum by week 4 (P450IA1, 0.2 nmol/mg; P450IA2, 0.8 nmol/mg; P450IIIA6, 0.12 nmol/mg) and decreased in the adult (P450IA1, 0.2 nmol/mg; P450IA2, 0.4 nmol/mg; P450IIIA6, 0.09 nmol/mg). Cytochromes P450IA1 and P450IA2 were not expressed in the untreated fetus. Onset of CYP3A6 gene expression occurred at day 30 of gestation and both transcription and mRNA accumulation were transplacentally inducible by rifampicin only shortly before birth, i.e. after treatment of the females between days 28 and 30 of gestation. Both long (1.85 kb) and short (1.7 kb) mRNA transcripts were expressed in untreated or rifampicin-treated fetuses. CYP3A6 gene expression was also induced by rifampicin in 1-week-old and 2-week-old animals. Developmental expression of CYP1A1 and CYP1A2 genes was shown to be closely related to the diet change accompanying weaning which occurs at weeks 3-4. In animals subjected to either delayed (week 6) or early (week 2) weaning, sharp accumulation of messages, proteins and related activities were delayed or anticipated accordingly with respect to normal weaning. Artificially scheduled weaning gave similar results when repeated with biological-grade lucern (grown in the absence of chemical fertilizers, pesticides, etc.), the main constituent of commercial rabbit chow. While CYP3A6 gene expression could be brought forward by early weaning at week 2, both message and protein did not exhibit increased accumulation after delayed weaning at week 6, and remained at the low level of the new born animal. Treatment of 1-week-old and 2-week-old animals with triiodothyronine or of 3-week-old animals with propylthiouracil, an antithyroid factor, did not modify the normal pattern of developmental expression of genes CYP1A1, CYP1A2 and CYP3A6. It is concluded that (a) the onset of CYP3A6 gene expression in the fetus occurs at day 30 of gestation, (b) expression of this gene may be induced transplacentally by rifampicin, (c) CYP1A1, CYP1A2 and CYP3A6 gene expression is sharply activated at weaning, and (d) thyroid hormones appear not to be responsible for the pattern of developmental expression of these genes in the rabbit.

Aging↗
PubMed1991

Removal of N-linked oligosaccharides of presumptive ectoderm impairs neural induction in Pleurodeles waltl.

Studies were carried out on the embryo of the amphibian Pleurodeles waltl to investigate the potential role of the N-linked oligosaccharides of the ectodermal cell membrane in the neural induction process. Glycopeptidase F (GPase F) was used to cleave N-linked oligosaccharides on presumptive ectoderm. Removal of oligosaccharide moieties from ectoderm membrane glycoconjugates completely inhibited natural neural induction in vitro. On the other hand, Swainsonine (Sw) and 1-deoxynojirimycin (dNM), specific inhibitors of enzymes involved in glycosylation, provoked strong and persistent changes in the structure of the N-linked oligosaccharides of presumptive ectoderm but did not prevent neuralisation of treated ectoderm. We conclude that N-linked carbohydrates are implicated in the phenomenon of neural induction. However, the structural integrity of N-linked carbohydrates of target tissue is not itself critical in this process. The existence of specific carbohydrates on presumptive ectoderm was still questioned as receptors of neural signal.

1-Deoxynojirimycin↗
PubMed1990

Metabolic activation of the new tricyclic antidepressant tianeptine by human liver cytochrome P450.

Incubation of [14C]tianeptine (0.5 mM) with human liver microsomes and a NADPH-generating system resulted in the in vitro covalent binding of a tianeptine metabolite to microsomal proteins. This covalent binding required oxygen and NADPH. It was decreased by piperonyl butoxide (4 mM) by 81%, and SKF 525-A (4 mM) by 87%, two relatively non-specific inhibitors of cytochrome P450, and by glutathione (4 mM) by 70%, a nucleophile. Covalent binding was decreased by 54% in the presence of troleandomycin (0.1 mM), a specific inhibitor of the glucocorticoid-inducible cytochrome P450 IIIA3, but remained unchanged in the presence of quinidine (0.1 mM) or dextromethorphan (0.1 mM), two inhibitors of cytochrome P450 IID6. Preincubation with IgG antibodies directed against cytochrome P450 IIIA3 decreased covalent binding by 65% whereas either preimmune IgG or IgG antibodies directed against P450 IA1, an isoenzyme inducible by polycyclic aromatic compounds, exhibited no significant inhibitory effect. We conclude that tianeptine is activated by human liver cytochrome P450 into a reactive metabolite. This activation is mediated in part by glucocorticoid-inducible isoenzymes but not by P450 IID6 (the isoenzyme which oxidizes debrisoquine) nor by P450 IA1 (an isoenzyme inducible by polycyclic aromatic compounds). The predictive value of this study regarding possible idiosyncratic and immunoallergic reactions in humans remains unknown.

Antidepressive Agents, Tricyclic↗
PubMed1990

Purification of a sheep liver cytochrome P-450 from the P450IIIA gene subfamily. Its contribution to the N-dealkylation of veterinary drugs.

Oral administration of troleandomycin at a dose of 100 mg/kg/day for 6 days to three adult male Lacaune sheep produced a 1.6-fold increase in specific content of liver microsomal cytochrome P-450. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, microsomal preparations from treated animals exhibited a strong band in the zone of electrophoretic mobility of cytochromes P-450. This band corresponded to a cytochrome P-450 which cross-reacted with rabbit P450IIIA6 antibodies, as demonstrated by immunoblotting. The ovine isozyme was purified to electrophoretic homogeneity by means of successive DEAE cellulose, CM cellulose and hydroxylapatite chromatographic separations. This hemoprotein had an apparent molecular weight of 52 kD as determined by calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was characterized in terms of spectral data, NH2-terminal amino acid sequence, immunologic and catalytic properties. This study revealed some interspecies differences with the orthologous rabbit isozyme. The contribution of this form to the N-demethylation of erythromycin and of three veterinary drugs: chlorpromazine, chlorpheniramine and bromhexine was demonstrated from inhibition by TAO, from immunoinhibition studies, using polyclonal antibodies raised in rabbit and from the existence of significant correlations between its microsomal level and these N-demethylase activities. In contrast, the results suggest that ovine P450IIIA could not be predominantly involved in the N-dealkylation of benzphetamine, ephedrine, ivermectine or spiramycin.

Administration, Oral↗
PubMed1990

Ontogenesis of rabbit liver cytochrome P450. Evidence for a cytochrome P450-IIE (3a)-related form prevailing during the post-natal period.

The liver hydroxylating system, mainly composed of cytochromes P450, is not highly active during foetal life. If develops after birth and reaches the adult level several weeks post-partum. We have studied the ontogenesis of rabbit cytochrome P450 during the post-natal period. Total P450 as well as isozymes 2, 3b, 3c, 4 and 6 were measured. The evolution of these proteins with ageing, together with qualitative modification of an electrophoretic profile, produced evidence of an early developing P450 prevailing from one week to three weeks after birth. We isolated and characterized a cytochrome, called P450 2y, from two-week liver microsomes. It is closely related to P450 3a, an adult form of rabbit P450 induced by ethanol. They have similar molecular masses, the same lambda max of CO-reduced spectrum and exhibit immunological cross-reactivity. However, we cannot conclude that the two proteins are identical from N-terminal amino acid analysis or the two-dimensional gel electrophoresis pattern. These results, as well as the recent evidence of two different genes coding for the P450 3a family, strengthen the idea that P450 2y and 3a are distinct proteins. P450 2y seems to be an early developing form abundant soon after birth, while P450 3a is a delayed form appearing like most P450 isozymes during the fourth post-natal week. Besides the quantitative development during perinatal life, there is an important qualitative modification of liver cytochrome P450 content.

Aging↗
PubMed1990

Omeprazole is an aryl hydrocarbon-like inducer of human hepatic cytochrome P450.

Omeprazole is a new drug used for its high efficiency as an inhibitor of gastric acid secretion. This substituted benzimidazole molecule had been shown to decrease several liver cytochrome P450-mediated monooxygenase activities both in vitro and in vivo. The present work was undertaken to determine whether this drug was an inducer of cytochrome P450 in humans. Primary cultures of human hepatocytes were maintained in a serum-free, chemically defined medium for 0-96 hours in the absence or in the presence of omeprazole (1-100 mumol/L) or of other cytochrome P450 inducers such as 3-methylcholanthrene, beta-naphthoflavone, or rifampicin for comparison. Omeprazole produced a time- and concentration-dependent increase in (a) cytochrome P450IA2 accumulation determined by western blot in microsomes from omeprazole-treated cells, while the level of other cytochrome P450 forms including P450IID6, IIE1, and IIIA was not increased in the same culture; (b) several monoxygenase activities, including phenacetin deethylase and acetanilide hydroxylase (cytochrome P450IA2) and ethoxyresorufin deethylase and benzpyrene hydroxylase (cytochrome P450IA1); (c) cytochrome P450IA2 de novo synthesis, determined by immunoprecipitation of cell lysate from [3H]Leu-labeled cells; (d) cytochromes P450IA1 and IA2 mRNAs, determined by northern blot analysis. An in vivo study was carried out on liver microsomes from five patients for whom hepatic biopsy specimens were available before and after repeated administration of omeprazole (20 mg/day for 4 days). In all cases, several-fold increases in cytochrome P450IA2 and specific cytochrome P450IA subfamily-dependent monooxygenase activities were observed in agreement with the results from cell culture. It was concluded that omeprazole is an aryl hydrocarbon-like inducer of cytochrome P450 secretion in human liver both in vitro and in vivo. This drug is therefore likely to increase the metabolism of any xenobiotic specifically oxidized by a cytochrome P450IA subfamily. This could potentiate the hepatotoxicity of phenacetin or paracetamol and activation of procarcinogens.

Aged↗
PubMed1989

Metabolic activation of the antidepressant tianeptine. I. Cytochrome P-450-mediated in vitro covalent binding.

Incubation under air of [14C]tianeptine (0.5 mM) with a NADPH-generating system and hamster, mouse or rat liver microsomes resulted in the in vitro covalent binding of [14C]tianeptine metabolites to microsomal proteins. Covalent binding to hamster liver microsomes required NADPH and oxygen; it was decreased in the presence of the cytochrome P-450 inhibitors, carbon monoxide, piperonyl butoxide (4 mM), and SKF 525-A (4 mM) or in the presence of the nucleophile, glutathione (1 or 4 mM). In vitro covalent binding to hamster liver microsomes was not decreased in the presence of quinidine (1 microM), and was similar with microsomes from either female Dark Agouti, or female Sprague-Dawley rats. In contrast, in vitro covalent binding to hamster liver microsomes was decreased in the presence of troleandomycin (0.25 mM), while covalent binding was increased with microsomes from either hamsters, mice or rats pretreated with dexamethasone. Preincubation with IgG antibodies directed against rabbit liver glucocorticoid-inducible cytochrome P-450 3c(P-450 IIIA4) decreased in vitro covalent binding by 53 and 89%, respectively, with microsomes from control hamsters and dexamethasone-pretreated hamsters, and by 60 and 81%, respectively, with microsomes from control and dexamethasone-pretreated rats. We conclude that tianeptine is activated by hamster, mouse and rat liver cytochrome P-450 into a reactive metabolite. Metabolic activation is mediated in part by glucocorticoid-inducible isoenzymes but not by the isoenzyme metabolizing debrisoquine. In vivo studies are reported in the accompanying paper.

Animals↗
PubMed1989

Electric-field-induced permeabilization and fusion of embryonic amphibian cells.

The technique of electropulsation has been shown to be highly efficient in promoting penetration of exogenous molecules into living cells, transfection, and cell fusion in different animal, vegetal, and bacterial cell systems. Introduction of such exogenous compounds, i.e., plasmids, into living cells is of great interest for embryological studies. Embryonic amphibian ectodermal cells from Pleurodeles waltl gastrulae, either freshly dissociated or cultured for 5 days, can be permeabilized when submitted to an external electric field of sufficient intensity: 500 V/cm for isolated spherical cells and 150-200 V/cm for plated cultured cells. Permeabilization was indicated by both the leakage of metabolites (ATP) from the cells and the uptake of exogenous compounds (pyranin) into the cells. With the use of higher field intensities (600 V/cm for freshly dissociated cells and 300 V/cm for cultured cells) cell fusion and syncytial structures could also be obtained. Isolated spherical cells had 100% viability immediately after being pulsed at intensities up to 600 V/cm. Cell lysis was observed above this value, although the nonlysed cells were observed to spread on a substrate and differentiate normally. For the cultured plated cells, cell viability fell with increasing electric-field strength, and for a given electric field value, cell viability decreased with the age of the culture after pulsing. Nevertheless, for electric-field intensities less than or equal to 300 V/cm, 100% of the cells remained attached to the substrate and differentiated normally over the following 5 days.

Adenosine Triphosphate↗
PubMed1989

Rat liver microsomal progesterone metabolism: evidence for differential troleandomycin and pregnenolone 16 alpha-carbonitrile inductive effects in the cytochrome P-450 III family.

The effect of Troleandomycin (TAO) and pregnenolone 16 alpha-carbonitrile (PCN) on the hepatic microsomal progesterone metabolism in the rat is evaluated. Over thirteen hydroxylated progesterone derivatives are detected, including the novel 6 beta, 21-, 6 beta, 16 alpha-, 6 beta, 16 beta- and 2,21-dihydroxy derivatives, suggesting the induction of several cytochrome P-450 isozymes. PCN treatment results overall in an augmented production of progesterone metabolites whereas TAO treatment both induces and represses specific hydroxylase activities. Progesterone metabolism with purified isozymes isolated from liver microsomes from TAO and PCN treated rats differs significantly from that observed with intact microsomes, reflecting the complexity of the induction pattern of the cytochrome P-450 III family.

Animals↗
PubMed1989

The increase in urinary excretion of 6 beta-hydroxycortisol as a marker of human hepatic cytochrome P450IIIA induction.

1. Urinary excretion of 6 beta-hydroxycortisol, hepatic microsomal cortisol 6 beta-hydroxylase and the specific content of several forms of cytochrome P450 were measured in 8 to 14 patients before and after treatment with rifampicin (600 mg orally per day for 4 days). 2. Rifampicin treatment produced an average five fold increase in daily excretion of urinary 6 beta-hydroxycortisol. 3. Cortisol 6 beta-hydroxylase activity increased from 15 +/- 6 pmol min-1 mg-1 in organ donors (considered as 'control subjects') to 87 +/- 31 pmol min-1 mg-1 in rifampicin treated patients. 4. Among three forms of human P450 (P450IA, IIC and IIIA), (1), (2), measured by Western blots, only P450IIIA was significantly induced by the antibiotic. 5. Only antibodies against P450IIIA selectively inhibited cortisol 6 beta-hydroxylase in human liver microsomes. 6. Cortisol 6 beta-hydroxylase was correlated with P450IIIA specific content. 7. The urinary level of 6 beta-hydroxycortisol correlated with liver microsomal cortisol 6 beta-hydroxylase and P450IIIA specific content. 8. We conclude that P450IIIA is predominantly responsible for cortisol 6 beta-hydroxylase activity in human liver microsomes and that urinary 6 beta-hydroxycortisol is a marker of the induction of this cytochrome P450.

Adult↗
PubMed1988

Inhibition of cytochrome P-450p (P450IIIA1) gene expression during liver regeneration from two-thirds hepatectomy in the rat.

Regenerating liver from partial hepatectomy (HPX) is known to exhibit a strong and transient deficiency in both spectrally detectable microsomal cytochrome P-450 (P-450) and related monooxygenase activities. Male Wistar rats (250-300 g) were HPX or sham operated and liver was excised at different times after operation. The time course of accumulation of five different forms of P-450 (including P-450b/e, P-450c, P-450d, P-450p and P-450UT-A) was determined in the regenerating liver, by Western blots developed with specific antibodies. With the exception of P-450c, whose level was not affected, the accumulation of other forms strongly decreased during the first 24 hr after HPX. For P-450b/e and P-450d, 80% of initial level was restored at 96 hr, whereas for P-450p and P-450UT-A, two major forms in control rat liver, the accumulation was only 20-25% of the initial, 1 week after HPX. No significant decrease was observed in sham operated animals. Plasmid pDex 12 containing a cDNA insert coding for P-450p was used to further investigate the effects of HPX on P-450p mRNA level and gene transcription. Northern blot analysis of RNA from regenerating liver (cDNA insert of pDex 12 being used as a probe) demonstrated that P-450p mRNA level decreased strongly to a minimum 12 hr after operation. This was correlated with a strong and transient decrease in P-450p gene transcription determined from nuclear run on experiments, the time course of which, however, did not account for the early decrease in mRNA level. We conclude that P-450p deficiency in the regenerating liver results from a combination of transient inhibition of gene transcription and early increase of mRNA degradation. Time course and amplitude of the decrease in P-450 UT-A accumulation suggest an inhibition of gene transcription as observed with P-450p.

Animals↗