Effect of retinol and retinoic acid supplemented diets on cytochrome P-450 content and UDP glucuronosyltransferase activities in vitamin A-deficient rat liver.
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Animals↗
Biomedical subjects
A Guillouzo
Publications and source records attributed to A Guillouzo.
Effects of halothane on human and rat hepatocyte cultures.
The aim of this study was to investigate direct cytotoxicity to human and rat hepatocytes in primary culture from halothane and compare it with that of isoflurane, which is known to be minimally metabolized and less toxic in vivo. Both human and rat parenchymal cells were isolated by the two-step collagenase perfusion method and after attachment to plastic were incubated with either volatile anesthetic for 24 h. All the cultures were maintained in 20% O2 condition and were not induced prior to anesthetic treatment. Temperature, atmosphere conditions, and anesthetic concentrations were kept constant during the study period. Evaluation of cytotoxicity was based on morphologic, biologic (determination of both extracellular and intracellular lactate dehydrogenase activity), and metabolic (protein synthesis and secretion) end points. Protein synthesis and secretion rates were found to be the most sensitive parameters in hepatocyte cultures from both species. Protein synthesis was inhibited by 18% and protein secretion by 50% in the presence of 1 and 1.25 mM halothane, respectively, in human cell cultures (P less than 0.05). With 1.25 mM halothane intracellular lactate dehydrogenase was also decreased; lactate dehydrogenase leakage and morphologic alterations were detected only beyond 5 mM halothane. By contrast, in rat hepatocyte cultures protein secretion was inhibited by 26% and protein synthesis by 20% in the presence of 0.1 and 0.75 mM halothane, respectively, whereas morphologic alterations and a 37% lactate dehydrogenase leakage increase were observed with the concentration of 1 mM (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Differential expression of laminin chains and receptor (LBP-32) in fetal and neoplastic hepatocytes compared to normal adult hepatocytes in vivo and in culture.
Laminin deposition is increased in fetal liver and in a variety of liver diseases, including the development of carcinoma. We investigated the role of the hepatocyte in the synthesis of both laminin and its 32- to 67-kd receptor in normal adult, fetal, and diethylnitrosamine-treated rat livers, and in adult rat hepatocyte primary cultures. Laminin was localized by immunoelectron microscopy in the endoplasmic reticulum of hepatocytes in fetal-derived and in 18-month-old diethylnitrosamine-treated rat livers. Steady-state mRNA levels for the three chains of laminin (A, B1, and B2) and the laminin receptor (LBP-32) were examined. Northern-blot analyses showed that hepatocytes at all stages lacked the A-chain mRNA. B1-chain mRNA was undetectable in normal adult hepatocytes, while significant levels of B1-chain mRNA were found in fetal hepatocytes and in adult hepatocyte primary culture. In hepatocytes from diethylnitrosamine-treated rats, B1-chain mRNAs were abundant and were present mainly in nodular formations rather than in the nontumorous areas. B2-chain mRNAs were barely detectable in either normal adult or fetal hepatocytes. In diethylnitrosamine-treated rats, the steady-state B2-chain mRNA level was higher in nodules than in nontumorous areas. In primary culture, B2-chain mRNAs were present as early as 4 hours after adult hepatocyte seeding, and dramatically increased during the following 2 days. Only low levels of laminin-receptor (LBP-32) mRNAs were present in normal adult hepatocytes, whereas the levels were high in the fetal and in the tumor-containing livers. In diethylnitrosamine-treated rats, LBP-32 mRNAs were more abundant in nodular formations rather than in nontumorous areas. In hepatocyte primary culture, the expression of the LBP-32 mRNA dramatically increased during the first 24 hours. These results show that in hepatocytes, expression of laminin chains and its receptor LBP-32 are not coordinated and depends on the maturation of the cells. In addition, they suggest that the expression of B1 and B2 chains in adult hepatocytes is related to changes of the normal phenotype and/or the pericellular environment.
Animals↗
[Cultured hepatocytes and drugs].
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Animals↗
Human liver fibroblast capacity for synthesizing interstitial collagenase in vitro.
si e s------------------------ABSTRACT------------------------- P4 initiates specifically the degradation of interstitial collagen types I-III. This enzyme is thus directly involved in the remodeling of the connective matrix. Fibroblasts are considered as the major source of interstitial collagenase in many tissues. However previous studies shown that liver fibroblasts did not spontaneously produce the enzyme. We have thus measured the steady-state levels of mRNA for interstitial procollagenase and quantified interstitial collagenase activity in cultured human liver fibroblasts, in presence or not of interleukin-1 or tumor necrosis factor. We demonstrate that human liver fibroblasts have the capacity for producing interstitial collagenase and that this production is regulated at a transcriptional step. We suggest that the liver fibroblast could represent a key cell for therapeutic strategies of fibrosis reversal.
Collagenases↗
Reversibility of hepatic fibrosis in experimentally induced cholestasis in rat.
The reversibility of hepatic fibrosis was investigated in an experimental model of extrahepatic cholestasis in the rat after common bile duct ligation for 2 weeks, followed by bilioduodenal anastomosis for 3 weeks. Bile duct ligation resulted in a transitory marked elevation in the serum concentration of 5'-nucleotidase, alkaline phosphatase, and bilirubin during the first 3 days. Then these levels decreased to threefold, twofold, and 100-fold the normal values, respectively, during the following 4 weeks. Histologic examination of the liver disclosed extensive bile duct proliferation and the formation of periportal fibrosis, with only slight inflammation and necrosis. The distribution of the major components of the hepatic extracellular matrix was analyzed 2 weeks after bile duct ligation, using the indirect immunoperoxidase method. Fibrous septa were found to be strongly stained for collagens I, pro-III, III and IV, fibronectin, and laminin. The most intense staining was found in enlarged periportal areas, collagen IV and laminin being particularly abundant around newly formed bile ducts. These changes paralleled high steady-state levels of alpha 1(I) and alpha 1(IV) collagen and B2 chain laminin mRNAs. Relief of the obstruction for 2 weeks resulted in a shift in the serum concentration of 5'-nucleotidase, alkaline phosphatase, and bilirubin toward normal values. A dramatic resorption of bile duct proliferations and periportal fibrosis were observed. Three weeks after bile duct repermeabilization, immunohistochemical study showed that the pattern of distribution of extracellular matrix components was almost normal, except for collagen IV, which remained abundant in the sinusoids when compared with the normal liver. In parallel, the steady-state B2-chain laminin mRNA level became lower than in cholestatic livers, whereas alpha 1(I) and alpha 1(IV) mRNAs were almost undetectable. These results show that hepatic fibrosis induced by experimental extrahepatic cholestasis in rat disappears in less than 3 weeks after relief of bile duct obstruction, suggesting that an active degradation of matrix protein occurs, except for collagen IV in the sinusoid.
Animals↗
Ultrastructural localization of the major components of the extracellular matrix in normal mouse liver.
We have investigated the extracellular and intracellular distribution of the major components of the hepatic extracellular matrix: types I and IV collagen (CI and CIV), type III procollagen (PCIII), fibronectin (FN) and laminin (LM) in the normal mouse liver by electron microscopy, using the indirect immunoperoxidase method. Extracellular localization of CI and PCIII revealed the typical collagen fibrils and small bundles in portal tracts and Disse's space. Faint extracellular deposition of CIV was detectable in basement membranes of portal structures and as discontinuous precipitates in Disse's space. FN strongly reacted in Disse's space and formed a continuous layer as well as strands between hepatocytes and endothelial cells. Moderate LM reaction was seen along the sinusoids in discrete spots and in basement membranes surrounding bile ducts and blood vessels. The cellular source of these components was demonstrated to be of mixed origin. Hepatocytes synthesized very little CI and large amounts of FN. Additionally, CI was produced by periportal fibroblasts. Fat-storing cells clearly participated in the production of PCIII, CIV and LM. LM synthesis was also found in bile duct cells and the cells of Hering's canal.
Animals↗
Changes in expression of mRNA coding for glutathione S-transferase subunits 1-2 and 7 in cultured rat hepatocytes.
mRNA hybridizing to probes for glutathione S-transferase (GST) subunits 1 and 2 (probe pGSTr 155) and subunit 7 (probe pGSTr 7) has been measured by Northern blot analysis in adult rat hepatocytes both in conventional monoculture and in co-culture with epithelial cells. In addition, several media conditions were used, namely with and without fetal calf serum (FCS) and with and without nicotinamide or dimethyl sulfoxide (DMSO). In monoculture, mRNA coding for subunits 1 and 2 was extensively reduced in the presence of FCS. In the absence of FCS, after an initial decrease, an increase of subunits 1 and 2 mRNA was noticed on day 6. When nicotinamide or DMSO was added to the medium, the GST subunits 1 and 2 mRNA level increased during the culture period. In co-culture, an initial reduction in levels of mRNA encoding subunits 1 and 2 was less marked and the values measured increased with co-culture time. Nicotinamide tended to reduce these mRNA levels, whereas DMSO increased them. In contrast, in conventional culture, mRNA encoding subunit 7 was expressed de novo and this induction was prevented by DMSO but not by nicotinamide. Similar results were obtained with co-culture.
Animals↗
Regulation of glutathione S-transferase subunits 3 and 4 in cultured rat hepatocytes.
mRNA levels of glutathione S-transferase (GST) subunits 3 and 4 were measured with a specific cDNA probe in adult rat hepatocytes maintained either in conventional culture or in coculture with rat liver epithelial cells. Four media conditions were used, i.e. with or without fetal calf serum (FCS) and with nicotinamide or dimethylsulfoxide (DMSO). When FCS was present in the culture medium, GST subunit 3 and 4 mRNAs were expressed at a level close to that found in freshly isolated hepatocytes during the whole culture period both in conventional culture and in coculture. All other culture conditions resulted in an increase of GST 3 and 4 mRNA levels. After exposure to phenobarbital an increase in GST 3 and 4 mRNA levels was demonstrated in both culture systems. Comparison with previous findings on the expression of GST subunits 1, 2 and 7 in the same culture conditions indicates that the different classes of GST are regulated independently.
Animals↗
Effect of phenobarbital on the expression of glutathione S-transferase isoenzymes in cultured rat hepatocytes.
Cultured adult rat hepatocytes were treated daily with 3.2 mM phenobarbital (PB) in order to study its effect on the expression of cytosolic glutathione S-transferase isoenzymes. Glutathione S-transferase (GST) activities, using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, were increased when PB was present in the culture medium. After purification and separation of GST on glutathione Sepharose 6 B and reversed-phase HPLC, respectively, it was observed in vitro that PB caused an increase in the relative amounts of subunits 1, 3 and 7 compared to subunits 2 and 4. Using Northern blot technique, elevated levels of GST subunit 1/2 and 7 mRNA were measured, after addition of PB to the cultures.
Animals↗
Participation of hepatocytes in the production of basement membrane components in human and rat liver during the perinatal period.
Little is known about the role of the extracellular matrix in cellular growth, migration and differentiation in the developing liver. The distribution and origin of the main constituents of the hepatic extracellular matrix have never been studied during liver differentiation. We have investigated the extracellular and intracellular distribution of fibronectin, laminin and types I, III and IV collagen in both rat and human liver during the perinatal period by light and electron microscopy, using the indirect immunoperoxidase method. All these components were demonstrated extracellularly, located mainly in portal spaces and, to a lesser extent, surrounding central veins. In perisinusoidal spaces, variations in distribution were observed depending on the matrix protein, the age of the donor and the species. In fetal rat liver, fibronectin formed a continuous layer around hepatocyte clusters while laminin and type III procollagen were present in small amounts. Collagens and laminin were visualized more easily in newborn rat liver. Fetal and newborn human liver contained higher amounts of matrix components than their rat counterparts. Fibronectin also reacted strongly in the sinusoid, and laminin and collagens formed discontinuous deposits. The source of this extracellular matrix was demonstrated to be of mixed origin. The major finding was the presence of immunoreactive laminin in the rough endoplasmic reticulum of hepatocytes irrespective of the age or species. In addition, hepatocytes contained large amounts of fibronectin and little of type I collagen. Another basement membrane component, type IV collagen, was also found in hepatocytes from all groups except fetal rat. Perisinusoidal cells also contained various matrix components including laminin, type III procollagen and, again with the exception of fetal rat liver, type IV collagen. The greater amounts of basement membrane components in the sinusoids of developing liver than in adult tissue and the participation of immature hepatocytes in the production of laminin and to a lesser degree of type IV collagen suggest that these matrix proteins play a critical role during liver differentiation.
Animals↗
Comparison of caffeine metabolism by slices, microsomes and hepatocyte cultures from adult human liver.
1. Caffeine was used as a molecular probe for hepatic monooxygenase activity in three in vitro models from human livers: slices, microsomes and hepatocyte cultures. 2. A h.p.l.c. method for determination of all possible metabolites of caffeine (16 compounds) is described. 3. Caffeine biotransformation by these three in vitro systems was low. However, metabolite formation was shown to proceed at a rate close to that calculated from in vivo caffeine elimination half-life. 4. Metabolic profiles were the same whatever the in vitro system used. The ratio of primary demethylated metabolites was similar to that measured by in vivo studies, i.e. the selectivity for caffeine N-3 demethylation to paraxanthine was retained. 5. Significant correlation (p less than 0.001) between 7-ethoxyresorufin O-deethylation and caffeine demethylations was demonstrated for the 12 human livers studied.
Adolescent↗
Effects of iron overload on transferrin secretion by cultured fetal rat hepatocytes.
Primary cultures of fetal rat hepatocytes were maintained in an arginine-free medium deprived of serum but supplemented with 0.03 mM L-ornithine and 10(-8) M dexamethasone. Ferric nitrilotriacetate was added to the culture medium in order to obtain iron concentrations ranging from 10 to 100 microM. After 24 h of treatment, iron was visualized inside the hepatocytes by the staining method of Perls and electron microscope study. The present data demonstrate that iron overload decreases transferrin secretion; this effect appears not to be specific, since albumin production is affected in a similar manner. This depressed transferrin secretion is not the consequence of hepatocyte death, as the phenomenon is confirmed when expressed per cell. Since the corresponding mRNA is unaffected, it may be postulated that iron overload decreases transferrin secretion at some post-transcriptional level.
Albumins↗
[Drugs and the culturing of hepatocytes].
Two model systems are described for studying isolated hepatocytes in vitro: pure culture for short-term designs and co-culture with epithelial rat liver cells (for long duration procedures). Acute or chronic liver toxicities are discussed as well as indirect toxicities. Examples of species differences and genetic polymorphism are shown. Data on drugs used for the treatment of liver diseases are also presented.
Cells, Cultured↗
Different cytotoxicity and metabolism of doxorubicin, daunorubicin, epirubicin, esorubicin and idarubicin in cultured human and rat hepatocytes.
Both cytotoxicity and metabolism of five anthracyclines, namely doxorubicin, daunorubicin, epirubicin, esorubicin and idarubicin, were investigated in primary cultures of both rat and human adult hepatocytes and, for comparison, in a rat liver epithelial cell line. Toxicity was assessed by morphological examination and measurement of lactate dehydrogenase leakage after 24 hr of treatment. The rank order of toxicity for both rat and human hepatocytes was esorubicin greater than doxorubicin = epirubicin greater than or equal to idarubicin greater than daunorubicin, and for rat epithelial cells: esorubicin greater than or equal to epirubicin greater than idarubicin = daunorubicin = doxorubicin. Human cells were around 2-fold less sensitive than rat hepatocytes to all anthracyclines. Anthracyclines and their metabolites were analyzed by HPLC. Differences in both the percentages and routes of metabolism were demonstrated between rat and human hepatocytes. The main metabolite was the 13-dihydro-derivative (-ol derivative) in both species from daunorubicin, idarubicin and esorubicin. Glucuronides of epirubicin and epirubicinol were found only in human hepatocytes. In addition, several unidentified metabolites were detected of esorubicin, idarubicin and daunorubicin in rat hepatocytes. In human hepatocytes, only one unknown metabolite from daunorubicin and doxorubicin was found to be formed by cells from a different donor. In spite of variations between individuals, human hepatocytes generally metabolized anthracyclines more actively than did rat hepatocytes. Rat liver epithelial cells were only able to convert daunorubicin and idarubicin, the two molecules which have the best affinity for the non-specific NADPH-dependent aldoketoreductase system. Three compounds (doxorubicin, epirubicin and esorubicin) were present in large amounts in the cells as the parent drug, another (idarubicin) as the 13-dihydro-derivative. This comparative study on cytotoxicity and metabolism of five anthracyclines in rat and human hepatocyte cultures emphasises species differences and the importance of this in vitro model system for further analysis of the metabolism and effect of anthracyclines.
Adolescent↗
Caffeine and theophylline metabolism in newborn and adult human hepatocytes; comparison with adult rat hepatocytes.
Cultured hepatocytes from newborn human (three samples), adult human (eight samples) and adult rat livers were used to study the metabolism of theophylline and caffeine, two drugs of which the metabolic pathways are known to be cytochrome P-450-dependent. Known metabolic pathways of caffeine in vivo were qualitatively maintained. However, only the primary metabolites were formed through oxidative N-demethylation giving theophylline, paraxanthine and theobromine and, through C-8 hydroxylation, giving 1,3,7-trimethyluric acid and a ring-opened compound the 6-amino-5[N-formylmethylamino]1,3-dimethyl uracil. The ratio of the three dimethylxanthine metabolites was dependent upon the species (human, rat), development stage (newborn, adult) and environmental factors. Similarly, theophylline was metabolized as in vivo by the demethylation pathway giving, preferentially, 3-methylxanthine and not 1-methylxanthine, and by a C-8 oxidation giving 1,3-dimethyluric acid. In newborn hepatocytes, all pathways were absent except the well-known methylation to caffeine. Moreover, such a methylation also occurred in adult human hepatocytes. This result was explained by the very low metabolic capacity of cultured cells, allowing the detection of only direct metabolites. Indeed, the overall biotransformation of both the methylxanthines by primary cultures of hepatocytes was remarkably weak, confirming previous studies with liver microsomal incubations. Thus the metabolism rate did not exceed about 30 nmoles/10(6) cells/24 hr in human adults, except for two subjects which were characterized by an extensive metabolism and a different metabolic profile. These two subjects were probably induced in vivo by environmental compounds. Both quantitative and qualitative data obtained from this study were roughly correlated with other in vivo and in vitro studies. Overall the experimental model of cultured human hepatocytes was shown to be capable of assessing the metabolic profile of two methylxanthines which is in agreement with the situation encountered in vivo. This example suggests that a breakthrough may be brought in new drugs development by the predictability from human hepatocyte culture model to the in vivo human situation.
Adult↗
Glutathione transferase isoenzymes in cultured rat hepatocytes.
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Animals↗
Essential fatty acid pattern of glycerolipids in rat hepatocytes in primary culture and in coculture with rat liver epithelial cells.
The linoleic acid content of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and triglyceride (TG) rapidly fell in rat hepatocytes in primary culture up to four days and in coculture with liver epithelial cells up to eight days. At the same time, the level of polyunsaturated fatty acids (PUFA), especially arachidonic acid, remained constant in PE, slightly decreased in PC and dropped in TG. There was no variation of the nonessential PUFA, 20:3n-9. Linoleic acid supplementation of cultures 24 hr before the harvest induced a rise in the linoleic acid level of the three lipid classes. Arachidonic acid remained constant in TG and only slightly decreased in PE and PC at day 4 of primary culture and day 8 of coculture. The level of 20:3n-9 increased in PE and PC and much more in TG. This net increase in the arachidonic acid and 20:3n-9 levels in TG could not be explained only by a transfer from the phospholipid pools of PUFA because the phospholipid content of hepatocytes and PUFA levels of phospholipids did not vary under linoleic supplementation. The low percentage of arachidonic acid in epithelial cells rules out any participation of these cells in the increase of arachidonic acid in supplemented cocultures. Triglycerides may act as a storage pool for plasma PUFA up to four days of primary culture and eight days of coculture. Besides, coculture seems more potent than primary culture to maintain the phospholipid level, to spare the essential PUFA in PE and to increase the TG synthesis in response to linoleic acid supplementation.
Animals↗