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A Guillouzo

Publications and source records attributed to A Guillouzo.

At least 127 records · Page 7Linked to original sources
PubMed1992

[Extracorporeal bio-artificial liver using isolated hepatocytes. An experimental study in the rat].

A new type of bioartificial liver using isolated hepatocytes immobilized in alginate beads was developed and its capacity of correcting the metabolic deficiency of bilirubin conjugation in Gunn rats was assessed. Hepatocytes were isolated from Sprague-Dawley rats using the in situ collagenase perfusion technique, and they were then immobilized in Calcium alginate beads. The capacity of these immobilized hepatocytes to conjugate in vitro bilirubin was checked as compared to monolayer hepatocyte culture. The bioartificial liver consisted in a cylindrical bioreactor containing either alginate beads and hepatocytes (test group), or only alginate beads (control group). Gunn rats were connected to this bioreactor through and extracorporeal circulation system, and "bile samples were collected" every hour. Bilirubin mono and biconjugates were dosed in the bile using the high-performance liquid chromatography technique. The viability of alginate immobilized hepatocytes, determined before and after each experiment, was stable at 75%. In the test group, the total conjugate concentration rapidly increased to reach a maximum value of 204 +/- 16 microns after 3 hours, while in the control group, there were only conjugate traces (1 micron). These results show that the bioartificial liver is an efficient means to temporarily correct genetic deficiency in Gunn rats. Such a system could be of therapeutic interest in case of acute hepatic insufficiency.

Animals↗
PubMed1992

Anti-liver microsomes autoantibodies and dihydralazine-induced hepatitis: specificity of autoantibodies and inductive capacity of the drug.

Anti-liver microsomes (anti-LM) autoantibodies in patients with dihydralazine-induced hepatitis were found to react specifically with cytochrome P4501A2 (P4501A2) but not with P4501A1 expressed in yeast and bacteria. These results were confirmed by immunoinhibition of methoxyresorufin-O-demethylase activity (supported by the P4501A subfamily); anti-LM antibodies more strongly inhibited this activity in yeast expressing P4501A2 than in yeast expressing P4501A1. Anti-LM were shown to be specific to the disease; in three cases, these autoantibodies were present at high titers during disease, whereas the titers decreased upon recovery and became undetectable a few months after recovery. Thus, there exists a time-dependent relationship between the disease and the autoantibodies, which does not prove that the autoantibodies are causative of the hepatitis; they might only be a marker. The inductive capacity of dihydralazine toward P450 was also studied. In rats treated in vivo and in human hepatocytes treated in vitro with dihydralazine, a 2-fold increase in P4501A2- and P4501A-supported monooxygenase activities was found. The levels of the other P450 isoforms tested were unchanged during treatment, both in vivo in rats and in vitro in cultures of human hepatocytes. In human hepatocytes, dihydralazine produced a dose-dependent increase in the level of P4501A up to 0.1 mM; induction of P4501A was less strong at 0.2 mM and disappeared at 0.5 mM. The same treatment did not change the level of P4503A4, taken as control. The strong heterogeneity in the expression of P4501A enzymes in human liver and the capacity of these enzymes for induction by dihydralazine and by other compounds might be predisposing factors in this autoimmune disease.

Animals↗
PubMed1991

Differential expression of laminin chains in hepatic lipocytes.

The lipocyte is an important source of laminin in the normal liver. We have investigated the expression of the 3 chains of laminin in isolated rat lipocytes. Both B1 and B2 chains, but not A, were found in medium from 5-day-old lipocyte primary cultures by immunoblotting and immunoprecipitation of 35S-labeled proteins after reducing SDS-polyacrylamide gel electrophoresis. An additional polypeptide of Mr = 380,000 was identified by immunoprecipitation. Under non-reducing conditions only one Mr = 900,000 band was revealed. High levels of B1 and B2 mRNAs were also demonstrated in 5-day-old cultured lipocytes while at the time of seeding, only B2 chain mRNAs were clearly detectable. A chain mRNA was constantly absent. These results suggest that lipocytes produce a variant form of laminin in primary culture and that the Mr = 380,000 polypeptide could be unrelated to the A chain of laminin.

Adipose Tissue↗
PubMed1991

Effects of ethanol and clofibrate on expression of cytochrome P-450 enzymes and epoxide hydrolase in cultures and cocultures of rat hepatocytes.

Cultured and cocultured rat hepatocytes were used to study the effects of ethanol and clofibrate on cytochrome P-450 (P-450) enzymes and epoxide hydrolase. We showed that in the presence of ethanol, clofibrate or both compounds, rat hepatocytes were able to express, after 3 days of pure culture, quantitatively and qualitatively reasonable levels of most cytochrome P-450 enzymes and epoxide hydrolase, compared to freshly isolated hepatocytes. However, ethanol induced the P-450IA subfamily, and clofibrate the P-450-IVA subfamily. In cocultures, after 6 days, most P-450 enzymes were still expressed while P-450IIC11 was completely lost. Ethanol and clofibrate had the same effect as in pure culture. These results show, by modifying culture medium conditions and cell-cell interactions, that it is possible to maintain reasonable xenobiotic-metabolizing-enzyme expression; however, these conditions have to be improved in order to preserve better P-450 expression. The mechanism of these effects and the inducibility of these systems remain to be elucidated by a study at molecular level.

Animals↗
PubMed1991

Effect of the aging process on the gender and phenobarbital dependent expression of glutathione S-transferase subunits in brown Norway rat liver.

The effect of age, gender and phenobarbital treatment on the hepatic cytosolic glutathione S-transferase subunit composition was studied in Brown Norway rats. Affinity chromatography followed by reversed phase HPLC was used in order to separate the various glutathione S-transferase subunits. Corresponding steady-state mRNA levels were measured by Northern Blot analysis using cDNA clones hybridizing to mRNA encoding glutathione S-transferase subunits 1/2, 3/4 and 7, respectively. In all the age groups studied (15, 25, 53, 99, 112 and 136 weeks) the total amount of glutathione S-transferase protein was in untreated rats significantly higher in males (132 micrograms/mg cytosolic protein) than in females (91 micrograms/mg cytosolic protein) and significant gender dependent differences in the subunit composition were demonstrated. Aging seemed to be of minor importance in untreated as well as in phenobarbital treated rats. Under control conditions, the subunit composition of male rats between 15 and 136 weeks old consisted of 28, 12, 11 and 49% of subunits 1, 2, 3 and 4 respectively and of female animals of the same age groups of 38, 26, 7 and 30%, respectively. In all the age groups studied phenobarbital administration (45 mg/kg body weight, i.p., once a day for 7 days) doubled total glutathione S-transferase protein in both genders and affected the subunit composition in a significant way, emphasizing the already existing differences between genders. Subunits 1, 2 and 3, especially, were increased in male rats in comparison to females resulting in the observation that levels of glutathione S-transferase subunits studied became higher in males than in their female counterparts. The HPLC results were confirmed by steady-state mRNA analysis. In untreated rats, higher levels of mRNA encoding glutathione S-transferase subunits 1/2 and 3/4 were present in male than in female livers. Phenobarbital treatment increased mRNA levels in both genders. Subunit 7 was never detected. These effects were demonstrated in both young and old rats.

Aging↗
PubMed1991

Regulation of glutathione S-transferase gene expression by phenobarbital in cultured adult rat hepatocytes.

Previous studies, by using Northern blotting analyses, showed that phenobarbital (PB) affects the steady-state mRNA levels of glutathione S-transferase (GST) subunits 1/2, 3/4 and 7 in both conventional cultures of adult rat hepatocytes and co-cultures, with rat liver epithelial cells [Vandenberghe et al., 1989, FEBS Lett. 251, 59-64; Morel et al., 1989, FEBS Lett. 258, 99-102]. To determine whether PB acts at the transcriptional level, nuclear 'run on' experiments using cDNA probes hybridizing to GST subunits 1/2, 3/4 and 7 mRNA were performed on purified nuclei isolated from control and PB treated hepatocytes seeded under conventional and co-culture conditions. Data from this study demonstrate that the increase in steady-state mRNA levels observed in both conventional culture and co-culture after 4 days PB exposure results from an increased transcriptional activity of the GST genes. However, a substantial increase in steady-state mRNA levels in the absence of a commensurate increase in transcriptional activity at 12 days of co-culture, indicates that the barbiturate has also a stabilizing effect in vitro on the GST mRNAs.

Animals↗
PubMed1991

In vitro assessment of stereoselective hepatic metabolism of disopyramide in humans: comparison with in vivo data.

Metabolism of disopyramide (DP) enantiomers has been investigated in primary cultures of adult human hepatocytes. Results were compared with in vivo data obtained from a previous pharmacokinetic study (Le Corre et al. Drug Metab. Dispos. 16:858-864 1988). Metabolism of DP enantiomers as a function of incubation time showed constant velocity over time. The intracellular/extracellular distribution of both DP and mono-N-desisopropyldisopyramide did not appear to be stereoselective. Metabolism of DP enantiomers as a function of substrate concentration followed a first order kinetics. The average fractions of (-)-(R)-DP and (+)-(S)-DP metabolized in vitro (4.7 +/- 2.7 and 7.1 +/- 4.2%, respectively, n = 4) were about 5-fold lower than the fractions metabolized in vivo (26.0 +/- 6.0 and 40.2 +/- 8.8%, respectively, n = 6). The stereoselective index [(+)-(S)/(-)-(R)] of the N-dealkylation pathway obtained in vitro (1.51 +/- 0.11, n = 4) was very close to the one obtained in vivo (1.55 +/- 0.10, n = 6). These results highlight the interest of hepatocyte cultures in the evaluation of drug metabolism and especially in the assessment of stereoselectivity.

Adult↗
PubMed1991

Cellular sources of matrix proteins in experimentally induced cholestatic rat liver.

Collagens (I, III, and IV), fibronectin, and laminin were localized using the indirect immunoperoxidase technique 14 days after bile duct ligation, i.e., when extensive fibrosis and numerous neoformed bile ducts were observed. Extensive fibrous septa in enlarged portal spaces were stained for collagens I, III and IV, fibronectin, and laminin. Collagen IV and laminin were abundant around proliferative bile ducts. In addition, collagen IV was nearly continuous in the sinusoids. At the ultrastructural level, antigens were localized in the endoplasmic reticulum of several liver cell types. In portal spaces, bile duct cells and cells that form the transitional canal of Hering were strongly labelled for basement membrane components, particularly laminin, but not for collagens I and III and fibronectin, which were abundant in fibroblast-like cells. Inside the lobule, only Ito cells and, to a lesser extent, endothelial cells contained collagens, fibronectin, and laminin. Ito cells were found to be heavily stained for collagens III and IV, and laminin. Except for fibronectin, which was always abundant, precursors of extracellular matrix proteins were only slightly detectable in the endoplasmic reticulum of some hepatocytes, particularly those located close to altered areas. This study demonstrates that experimental extrahepatic cholestasis in the rat induces periportal fibrosis and continuous deposition of collagen IV in the sinusoids. Several cell types participate in the formation of extracellular matrix components, particularly bile duct cells and Ito cells, with a possible involvement of hepatocytes, thus suggesting that cholestasis provokes changes in the pattern of matrix protein production in liver cells.

Animals↗
PubMed1991

Effects of the prolyl 4-hydroxylase proinhibitor HOE 077 on human and rat hepatocytes in primary culture.

Alterations in extracellular matrix occur in many chronic liver diseases leading to the formation of hepatic fibrosis. We have studied the effects of the putative hepatoselective fibrosuppressive compound HOE 077, a proinhibitor of prolyl 4-hydroxylase, on normal adult human and rat hepatocytes in primary culture. In human hepatocyte cultures, the cytotoxicity of HOE 077 was assessed after a 20-h treatment at concentrations ranging from 0.125 to 2 mg/ml of medium. No significant change was found in cell morphology, neutral red uptake, red oil staining, lactate dehydrogenase release, tetrazolium salt reduction, ethoxyresorufin O-deethylase activity and protein synthesis; however, HOE 077 slightly decreased DNA synthesis at 2 mg/ml. In rat hepatocyte cultures, the cytotoxicity of the compound was assessed by testing the same parameters after a daily exposure of cultures for 2 days or 4 days, at concentrations ranging from 0.25 to 4.5 mg/ml of medium. Whatever the concentration, the compound had no obvious morphological effect. However, hepatocytes were less spread at the concentration of 4.5 mg/ml. HOE 077 at 2 mg/ml slightly decreased neutral red uptake but was without obvious effect on protein synthesis after 2 days. By contrast, on day 4, protein synthesis was markedly reduced in hepatocyte cultures exposed to HOE 077 at 4.5 mg/ml. Hydroxyproline content determination in media from 4-day-old hepatocyte cultures incubated with HOE 077 at 0.5 to 4.5 mg/ml, showed a dose-dependent decrease in the hydroxyproline/proline ratio in acetic acid soluble material. By indirect immunoperoxidase, intracellular collagen IV was found to be inhibited in hepatocyte cultures after 4 days of exposure to 4.5 mg/ml HOE 077.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Metabolism of doxorubicin, daunorubicin and epirubicin in human and rat hepatoma cells.

The metabolism of three anthracyclines, namely daunorubicin, doxorubicin and epirubicin, was studied in two human (HepG2 and HBG2) and one rat (FAO) hepatoma cell lines. Both species and substrate differences in anthracycline metabolism were retained by these hepatoma cell lines with regard to the aldoketoreductase pathway. However, glucuronidation of epirubicin normally found in vivo and in human hepatocyte cultures was not recovered in human hepatoma cells, further indicating that these cells express only some of the liver functions. Nevertheless, these cell lines could be a suitable model to investigate the still poorly understood aldoketoreductase activity.

Animals↗
PubMed1991

Expression of laminin and its receptor LBP-32 in human and rat hepatoma cells.

Dramatic cellular changes that occur during hepatocarcinogenesis are associated with major alterations in extracellular matrix formation and in the relationships between cells and their microenvironment. We have studied the expression of laminin, the major noncollagenous glycoprotein of basement membrane, and the laminin receptor 32 kD laminin-binding protein in two rat (Faza 967 and HTC) and two human (HepG2 and HBGC2) hepatoma cell lines that express a variety of liver-specific functions. Laminin was found in the rough endoplasmic reticulum of these cells when the indirect immunoperoxidase method and electron microscopic examination were used. Radiolabeled laminin, immunoprecipitated from both media and cell extracts, was resolved by electrophoresis on sodium dodecyl sulfate gel in two major polypeptides that comigrated with the A and B subunits from Engelbreth-Holm-Swarm tumor laminin. Immunoblot analysis showed that the Mr = 400,000 polypeptide did not correspond to the A subunit of laminin. Northern blot analyses demonstrated large amounts of B1 and B2 mRNAs but no A chain mRNA. We conclude that the tumor cells produce the laminin B chains only. In contrast, normal adult hepatocytes from either man or rat lacked laminin mRNAs, whereas in 1-day primary culture, B chain mRNAs became detectable. The steady-state level of 32 kD laminin-binding protein mRNA was 10-fold and threefold higher in rat hepatoma cells than in freshly isolated and 1-day cultured normal rat hepatocytes, respectively. In human hepatocytes, the steady-state levels of 32 kD laminin-binding protein mRNAs varied depending on the donor and never reached the level of the human hepatoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Intralobular distribution and quantitation of cytochrome P-450 enzymes in human liver as a function of age.

We have used immunohistochemical, immunoblotting and messenger RNA blotting approaches to study the distribution and quantitation of three cytochrome P-450 enzymes, namely P-450 IA2, P-450 IIC and P-450 IIIA and, for comparison, epoxide hydrolase and NADPH-cytochrome P-450 reductase in human liver. Age-related changes in both the amounts and the intralobular distributions of these enzymes were demonstrated, and the enzymes differ in these regards: In fetal liver, P-450 IA2 and P-450 IIC were very low, when present at all, whereas P-450 IIIA, epoxide hydrolase and the reductase were already abundant and found in all the hepatocytes. During the postnatal period, P-450 IIC dramatically increased and was observed in all hepatocytes, the centrilobular ones being more intensely stained. P-450 IIIA was restricted to centrilobular and midzonal hepatocytes in normal adult liver. P-450 IA2 showed this same intralobular distribution; however, its presence was detected only several weeks or months after birth as judged both by immunohistochemical and immunoblotting techniques. Epoxide hydrolase and NADPH-cytochrome P-450 reductase were easily visualized in all hepatocytes regardless of the age of the donor; in child and adult livers, centrilobular hepatocytes were more intensely stained. Immunoreactive protein contents and corresponding messenger RNA levels correlated well with immunohistochemical observations. No major modification was seen in fibrotic liver, whereas both positive and negative cells were observed in cirrhotic liver nodules for all enzymes studied.

Aging↗
PubMed1990

Transforming growth-factor-beta (TGF-beta) inhibits albumin synthesis in normal human hepatocytes and in hepatoma HepG2 cells.

We explored the effect of transforming growth factor beta (TGF-beta), a cytokine that appears to play a central role in inflammatory events, on albumin expression by normal adult human hepatocytes and hepatoma cells. Addition of TGF-beta to primary human hepatocyte cultures resulted in a dramatic decrease in albumin accumulation and synthesis. This effect was dose-dependent, took place after a 48h incubation period and was maintained over 96h. TGF-beta-decreased albumin protein levels were associated with reduced albumin mRNA content. Actin mRNA levels were concomittantly increased. Comparable qualitative effects of TGF-beta were observed on human hepatoma HepG2 cells.

Adult↗
PubMed1990

Expression of cytochrome P-450 enzymes in cultured human hepatocytes.

Hepatocytes from adult and newborn humans were put into primary culture and exposed to phenobarbital, 3-methylcholanthrene, or rifampicin, three well-known inducers of cytochrome P-450 in animals. The expression of four cytochrome P-450 enzymes (or groups of enzymes, namely P-450 IIIA, P-450 IIC8/9/10, P-450 IIE1, and P-450 IA2) was investigated. These enzymes were found to remain expressed during the period of culture studied. Treatment with the inducers for three days resulted in different responses, depending upon the inducer and the enzyme. Phenobarbital and rifampicin increased P-450 IIC8/9/10 mRNA transcripts and the corresponding protein, while 3-methylcholanthrene was ineffective. Both P-450 IIIA mRNA and protein were strongly induced by rifampicin. All of the hepatocytes were found to synthesize P-450 IIIA in response to rifampicin, as shown by immunoperoxidase staining. P-450 IIIA expression was not affected by phenobarbital and was decreased by 3-methylcholanthrene. P-450s IA2 and IIE1 decreased to 25-50% of the initial level during these cultures. P-450 IA2 and ethoxyresorufin O-deethylase activity (which is a monooxygenase activity related to P-450 IA family) were increased only by 3-methylcholanthrene and did not respond to the other inducers. P-450 IIE1 was not induced by any of these compounds. P-450 IIC8/9/10 and P-450 IIIA mRNA levels were also measured in human hepatocytes from one newborn. P-450 IIC8/9/10 was barely expressed in freshly isolated cells but increased dramatically with time in culture. P-450 IIIA transcripts were abundant in both freshly isolated and cultured cells derived from a newborn. These results clearly demonstrate that human hepatocytes continue to express cytochrome P-450 enzymes and respond to inducers in culture. This model system provides a useful approach for investigating the effects of drugs on maturation and expression of drug-metabolizing enzymes in human liver.

Adult↗
PubMed1990

Acute-phase-response induction in rat hepatocytes co-cultured with rat liver epithelial cells.

The response of rat hepatocytes co-cultured with rat liver epithelial cells to conditioned medium (CM) from lipopolysaccharide (LPS)-activated monocytes was investigated by measuring the concentration of alpha 2-macroglobulin (alpha 2M), alpha 1-acid glycoprotein (AGP), albumin and transferrin, as well as the changes in glycosylation of alpha 1-acid glycoprotein. During an initial 8-day treatment with CM, concentrations of alpha 2M and AGP increased markedly over those of control culture, whereas concentrations of albumin and transferrin decreased. The glycosylation pattern of AGP indicated an important relative increase of the concanavalin A-strongly-reactive (SR) variant upon treatment. When CM addition to hepatocyte culture medium was stopped, the concentrations of the four proteins and the glycosylation pattern of AGP reverted to those of control cultures. Further addition (on day 15) to cultures of CM increased the concentration of alpha 2M and decreased albumin and transferrin concentrations. Although AGP concentrations did not increase above those of controls, the appearance of the SR variant was again stimulated by CM. These results show that, in co-culture, rat hepatocytes remain able to respond to repeated inflammatory stimuli.

Albumins↗
PubMed1990

Methylcholanthrene but not phenobarbital enhances caffeine and theophylline metabolism in cultured adult human hepatocytes.

Biotransformation of caffeine and theophylline and the effect of two well-known inducers of P-450 isozymes, namely phenobarbital (PB) and methylcholanthrene (3-MC) were studied in cultured hepatocytes from six human adult donors. Hepatocytes co-cultured with rat liver epithelial cells maintained a higher metabolic capacity than pure cultures. PB treatment of cultured hepatocytes for 3 days slightly increased the rate of caffeine metabolism 1.4 +/- 0.5-fold (N = 6) vs controls, and theophylline metabolism 1.2 +/- 0.4-fold (N = 6), whereas 3-MC treatment increased metabolism markedly 5.8 +/- 2.3- and 3.3 +/- 1.1-fold (N = 6) vs controls for caffeine and theophylline, respectively. Paraxanthine and theophylline formations from caffeine were the most induced by 3-MC. Their increase was significantly correlated (rs = 0.89, P less than 0.007) but not with TB formation, suggesting that at least two isozymes of the P-450IA family are involved in the first demethylations of caffeine. In addition, the N-1 demethylation of theophylline (mean increase of 554% vs controls) was not correlated with the N-1 demethylation of caffeine (mean to increase 247% vs controls) for the same donor after 3-MC treatment, suggesting that these two demethylations are mediated by a different P-450.

Adult↗
PubMed1990

Primary culture of adult rat hepatocytes after 48-hour preservation of the liver with cold UW solution.

Rat livers were perfused and stored for 48 hr in cold University of Wisconsin solution before dissociation by the two-step collagenase method. At that time, glycogen content was significantly reduced, but no obvious changes in albumin, beta-actin and aldolase B mRNAs and in glutathione levels were observed. Enzymatic perfusion yielded 280 +/- 30 x 10(6) viable hepatocytes vs. 520 +/- 40 x 10(6) viable hepatocytes from unstored organs. Cell viability determined by trypan blue exclusion was 74% and 90%, respectively. Hepatocytes from University of Wisconsin-preserved livers had a 29% reduced adenosine triphosphate content, but glutathione levels did not significantly differ from those found in unstored cells. When put into culture, hepatocytes formed typical monolayers of granular epithelial cells and did not exhibit alteration of their fine structure when compared with cells from unstored organs. After 24 and 48 hr, they showed variations in cytochrome P-450 content and ethoxyresorufin O-deethylase activity similar to those observed with unstored cells. By contrast, overall protein synthesis and albumin secretion rate were 40% and 30% lower, respectively. Hepatocytes from University of Wisconsin-preserved organs could be cryopreserved and further cultured as unstored cells. The University of Wisconsin solution was also used to preserve isolated hepatocytes. Viability of freshly isolated hepatocytes was decreased by only 10% after 48 hr of hypothermic liver storage when assayed by intracellular lactate dehydrogenase content. However, after 4 hr of storage, in contrast with hepatocytes preserved in L15 Leibovitz medium, the cells attached poorly to plastic and exhibited morphological alterations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗