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Biomedical subjects

A Guillouzo

Publications and source records attributed to A Guillouzo.

At least 163 records · Page 9Linked to original sources
PubMed1988

Influence of medium composition and culture conditions on glutathione S-transferase activity in adult rat hepatocytes during culture.

Glutathione S-transferase (GST) activity was measured in adult rat hepatocytes during either pure culture or coculture with another rat liver cell type in various media. Addition of nicotinamide, selenium, or dimethylsulfoxide, deprivation of cyst(e)ine and the use of two complex media were tested. Whatever the conditions used, after a constant decrease during the first 24 h, GST remained active over the whole culture period (1-2 wk). However, various patterns were observed: GST activity either remained relatively stable to approximately 50% of the initial value or showed a moderate or strong increase. The highest values were found in pure hepatocyte cultures maintained in the presence of nicotinamide or dimethylsulfoxide. Similar changes were observed using 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene as substrates for GST. Addition of 10(-4) M indomethacin resulted in 37 to 60% inhibition of enzyme activity. Thus, these results demonstrate that GST remained expressed during culture but its levels markedly varied depending on the medium composition and type and age of culture.

Animals↗
PubMed1988

Cryopreservation of isolated rat hepatocytes: a critical evaluation of freezing and thawing conditions.

Various parameters, including the nature and proportion of the constituents of the cryoprotective medium, the cooling rate, and the composition of the thawing medium, were evaluated for the cryopreservation of adult rat hepatocytes. The highest percentage of cells able to survive in culture was obtained by freezing in L15 medium containing 16% dimethyl sulfoxide, at a rate of 3 degrees C/min, and by adding 0.8 M glucose to the thawing medium. More than 50% of hepatocytes capable of attachment just after cell isolation kept this property after freezing and survived in primary culture. Dead cells could be eliminated before seeding by centrifugation on a Percoll layer. In culture, frozen cells exhibited a morphology similar to that of unfrozen cells and after 24 hr their protein secretion rate was reduced by only about 40%.

Animals↗
PubMed1988

Types I and IV procollagen gene expression in cultured rat hepatocytes.

The molecular mechanism involved in the expression of collagens by hepatocytes were investigated in both pure and co-culture with another rat liver epithelial cell type (RLEC). We measured the steady-state levels of mRNAs coding for pro alpha 1(I) and pro alpha 1(IV) chains by Northern analysis and by dot blotting, using specific recombinant cDNA probes. In freshly isolated hepatocytes, only small amounts of pro alpha 1(I) and pro alpha 1(IV) mRNAs were detected by dot-blot analysis. After 3 days in culture, the pro alpha 1(I) and pro alpha 1(IV) mRNA levels increased 2 to 5 times. The amount of pro alpha 1(IV) mRNAs was identical in hepatocyte cultured with RLECs while the pro alpha 1(I) mRNA level was 5 times that in pure hepatocyte culture. Hydrocortisone reduced pro alpha 1(I) mRNA in hepatocyte cultures, but had no effect on co-cultured cells. In both culture systems, this glucocorticoid did not act on the steady-state pro alpha 1(IV) mRNA level. Whatever the age and the type of culture (pure or mixed) RLECs exhibited the highest levels of pro alpha 1(I) and pro alpha 1(IV) mRNAs, which were reduced by hydrocortisone. These results show that procollagen gene expression by hepatocytes is not directly correlated with their functional state and that corticosteroids differently affect the expression of different collagen genes and collagen deposition.

Animals↗
PubMed1988

Differential response of primary cultures of human and rat hepatocytes to aflatoxin B1-induced cytotoxicity and protection by the hepatoprotective agent (+)-cyanidanol-3.

The acute hepatotoxicity induced by aflatoxin B1 (AFB1) and the potential protective effect of (+)-cyanidanol-3 (Catergen) were evaluated in both human and rat hepatocytes in primary cultures. AFB1-induced acute toxicity was visualized by light microscope observation and quantified by measurement of lactic dehydrogenase activity in the medium. Human hepatocytes were susceptible to AFB1-induced cytotoxicity but no evident relationship between the concentration of mycotoxin and the extent of cellular damage was established. (+)-Cyanidanol-3 was not toxic at concentrations up to 2 x 10(-3)M, but no obvious protective effect from AFB1-induced injury was evidenced in human cells. By contrast, rat hepatocytes responded in a dose-related manner to AFB1. (+)-Cyanidanol was toxic at 10(-3)M, but even at this concentration exerted a strong protective effect against AFB1-induced cytotoxicity. Such species differences suggest the existence of metabolic differences in both AFB1 and (+)-cyanidanol-3 activating and deactivating mechanisms.

Adolescent↗
PubMed1988

Identification of metabolic pathways of pindolol and fluperlapine in adult human hepatocyte cultures.

1. Adult human hepatocytes were obtained from a normal donor and a barbiturate-overdose fatality and maintained either in pure culture or in co-culture with rat liver epithelial cells. At varying times of culture, metabolic pathways of pindolol and fluperlapine, two drugs which are transformed at different rates and routes in man and rat, were determined after a 24 h incubation. 2. In both culture conditions, human hepatocytes remained able to form the major urinary metabolites of pindolol and fluperlapine, including oxidized products, glucuronide and sulphate conjugates over the whole period studied (i.e. 5 and 21 days in pure and co-culture respectively). 3. Fluperlapine which has the higher first pass in man and animals was found to be metabolized in vitro more quickly than pindolol. 4. Some qualitative and quantitative differences were found between normal and drug-altered human hepatocytes, indicating that in vivo environmental factors may greatly influence metabolic properties of these cells even after several days in culture.

Animals↗
PubMed1988

Use of hepatocyte cultures for preliminary metabolism and cytotoxicity studies of a new anti-hypertensive agent, oxaminozoline.

1. Adult rat hepatocytes co-cultured with rat liver epithelial cells were used to evaluate chronic cytotoxicity of a new alpha 2 agonist, oxaminozoline (S-3341-3) compared to that of clonidine. The same maximum non-toxic concentration (25 micrograms per ml of medium) was found for both drugs after a daily treatment for 12 days. 2. Oxaminozoline metabolism was analysed in short-term hepatocyte cultures. Four metabolites resulting from oxidation or hydrolysis of the parent drug were identified. Three of the metabolites were identical to those reported in vivo. The presence of an additional minor metabolite in culture may be due to the higher metabolic rate of the drug in this model system.

Animals↗
PubMed1988

Use of hepatocyte co-cultures in the assessment of drug toxicity from chronic exposure.

1. In rat hepatocyte cultures, clonidine and malotilate were cytotoxic at 170 microM but did not induce changes at lower concentrations during 24 h exposure. Amitriptyline induced cell injury at 20 microM but was ineffective at 2 microM. 2. In co-culture of rat or human hepatocytes with rat liver epithelial cells, 2 microM amitriptyline was cytotoxic after 7 days treatment whereas 70 to 100 microM clonidine or malotilate gave no significant effect. 3. These results suggest that co-cultured hepatocytes which retain their differentiated state for several days or weeks, represents a promising tool for studying hepatotoxicity from chronic treatment in vitro.

Amitriptyline↗
PubMed1988

Hepatotoxicity and molecular aspects of hepatocyte function in primary culture.

1. The application of primary cultures of hepatocytes in testing for hepatotoxicity of drugs is reviewed. 2. Hepatotoxicity results principally from the biotransformation of toxic agents. This process is very complex and specific and involves a powerful system of multigenic isozyme families for both phase I and phase II drug metabolizing reactions. Many of the isozymes are specifically expressed in the liver in relation to the maturation or differentiation state, and are specifically induced, possibly through a complex temporally programmed gene regulation. 3. This highly specific, coordinated, molecular regulation is difficult to maintain in vitro. Isolation of hepatocytes induces a prompt differential decline of liver-specific gene transcription, which leads to preferential loss of the most specific functions, including those of the drug metabolizing isozymes, whereas repair of cell damage remains active. 4. The use of serum-free, hormonally defined media stabilizes specific hepatic functions, but not transcriptional activity, for 4-5 days. Defined media retain active DNA replication but do not permit clonal growth of hepatocytes. Co-culturing hepatocytes with primitive biliary cells prolongs cell survival and their functional capacities for several weeks, including some of the transcriptional activity.

Animals↗
PubMed1988

Iron chelators: in vitro inhibitory effect on the liver stage of rodent and human malaria.

The activity of desferrioxamine (Desferal) and desferrithiocin (a newly developed oral iron chelator) was evaluated against the liver stage of Plasmodium yoelii and P. falciparum in the rodent and the human hepatocyte in vitro culture system. The two iron chelators were found to inhibit the liver schizogony of both the rodent and the human Plasmodium species at concentrations achievable in vivo. P. falciparum proved to be more sensitive (ic 95% below 20 micromol/l than P. yoelii (ic 95% 50-100 micromol/l). As assessed by electron microscopy, drug administration was associated with focal clarification of the cytoplasm thought to be reversible. As desferrioxamine and desferrithiocin are known to be equally active on the blood stage of rodent and human plasmodia, iron chelators are deserving of further investigation as potential alternative candidates to existing drugs for radical cure of malaria.

Animals↗
PubMed1987

Modulation of alkaline phosphodiesterase I in cultured rat hepatocytes.

Alkaline phosphodiesterase I activity was measured in adult and foetal rat hepatocytes maintained in primary culture under various conditions. This enzyme was found to be expressed in both cell populations and could be resolved into two bands having apparent molecular weights of 130,000 and 250,000, respectively. Alkaline phosphodiesterase I activity was already at high levels in 15 day foetal liver and, as early as the 19th day of gestation, it reached adult levels. Alkaline phosphodiesterase I levels were well maintained during culture. In the absence of serum, its level continued to increase with time in foetal cells. It dramatically increased by days 4 and 5, in adult cells maintained on fibronectin and plastic, respectively. Dexamethasone stimulated alkaline phosphodiesterase I activity after a lag phase of 8 h, with a maximum reached after 40 h. As this induction was prevented by addition of actinomycin D or cycloheximide, it could be concluded that it required RNA and protein synthesis. Only the major Mr 250,000 form responded to dexamethasone and was sensitive to serum.

Age Factors↗
PubMed1987

Species differences in metabolism of ketotifen in rat, rabbit and man: demonstration of similar pathways in vivo and in cultured hepatocytes.

In vitro drug metabolism by cultured rat, rabbit and human adult hepatocytes has been studied, using ketotifen (ZADITEN) as a model substrate because it is biotransformed in vivo by various metabolic pathways in man and animals. The major in vivo pathways were demonstrated in vitro, namely oxidation in rat hepatocytes, oxidation, glucuronidation and sulfation in rabbit hepatocytes, reduction and glucuronidation in human hepatocytes. Human hepatocytes were the most stable in culture, displaying ketotifen biotransformation for at least one week. These results clearly demonstrated that cultured hepatocytes retain their in vivo specific drug metabolizing activities, including inter-species polymorphism, for a few days. Therefore, pure hepatocyte cultures represent a useful system suitable for drug metabolism studies.

Adult↗
PubMed1987

Chronic liver iron overload in the baboon by ferric nitrilotriacetate. Morphologic and functional changes with special reference to collagen synthesis enzymes.

Two baboons receiving intramuscular injections of ferric nitrilotriacetate over a two-year period were compared with two control baboons. The results indicate that in iron-overloaded animals: liver iron excess was major (maximal liver iron concentration values of 42 mumol/100 mg dry weight for both animals vs 1.3 +/- 0.2 (mean +/- SD) in controls) and chronic (for 15 months liver iron concentrations were higher than 15); iron deposition, although less abundant than in sinusoidal cells, was pronounced within parenchymal cells; serum transaminase activities were markedly increased; rare foci of perisinusoidal fibrosis were observed in areas of massive iron overload; and a dramatic decrease in hepatic 4-prolyl-hydroxylase activity was found, in contrast with unchanged glucosyltransferase and galactosyltransferase activities. In conclusion these findings suggest that, in our model, chronic liver iron overload: exerts a marked biochemical cytolytic effect; and does not produce significant hepatic fibrosis, possibly related to an inhibiting effect of ferric nitrilotriacetate complex on 4-prolyl-hydroxylase activity.

Animals↗
PubMed1987

Detection of mRNAs present at low concentrations in rat liver by in situ hybridization: application to the study of metabolic regulation and azo dye hepatocarcinogenesis.

In situ hybridization on tissue sections was used to detect mRNAs present at low concentrations during metabolic adaptation and azo dye carcinogenesis in rat liver. The method consisted of hybridizing the slices at relatively high stringency with [35S]-labeled single-stranded probes derived from cDNA insert clones into the M13 phage. L-pyruvate kinase mRNA was proved to be present at very low concentrations in hepatocytes of fasted rats and to be relatively abundant in all hepatocytes after 18 hr of refeeding on a carbohydrate-rich diet. Aldolase A mRNA concentrations have been previously shown to increase markedly in liver of 3'-methyl DAB-fed rats, with a maximum at the fourth week. We demonstrate here, using our in situ hybridization technique, that this phenomenon is not due to re-expression of this "fetal marker" in hepatocytes but to its abundancy in proliferating small cells (i.e., so-called oval and transitional cells). Small amounts were also detected in sinusoidal cells. In normal liver, aldolase A mRNAs were detected only in some sinusoidal cells.

Animals↗
PubMed1986

Essential fatty acid uptake and esterification in primary culture of rat hepatocytes.

Primary cultures of adult rat hepatocytes were used to compare the uptake and esterification of essential polyunsaturated fatty acids (18:2, 20:3 and 20:4 of the n-6 series) with those of palmitic and oleic acids. The uptake of unesterified fatty acids was linearly related to the free fatty acid/albumin molar ratio for 14 h and did not depend on the unbound free fatty acid level. Whatever the initial free fatty acid/albumin molar ratio, it dropped to 0.5 +/- 0.1 mM after 14 h, thus showing that hepatocytes have a high capacity for clearing free fatty acids from the medium at high free fatty acid/albumin molar ratios. The free fatty acid uptake become saturable when the free fatty acid and albumin concentrations were raised and the free fatty acid/albumin ratio remained constant. This strongly suggests that albumin-hepatocyte interaction mediates free fatty acid uptake. This uptake was identical whatever the fatty acid tested and did not depend on the relative amounts of fatty acids when they were added simultaneously. Triacylglycerol accumulation and synthesis, monitored by labelled fatty acids, were related to the free fatty acid/albumin molar ratio and exhibited no specificity for the series of fatty acids tested. Triacylglycerols were enriched in all the fatty acids tested by up to 60%, and fatty acid incorporation into diacylglycerols and triacylglycerols reflected the free fatty acid composition of the medium. By contrast, neither the level nor the synthesis of phospholipids varied with free fatty acid/albumin, but the rate of phospholipid turnover depended on the fatty acids tested. Accumulation of these acids was smaller in phospholipids than in triacylglycerols. When linoleic and arachidonic acids were added together, phospholipids (especially phosphatidylethanolamine and phosphatidylinositol) were more enriched in arachidonic acid than triacylglycerols. This might be due to the specificity for fatty acid of the enzymes involved in phospholipid metabolism.

Animals↗
PubMed1986

Immunocytochemical evidence for the maintenance of cytochrome P-450 isozymes, NADPH cytochrome C reductase, and epoxide hydrolase in pure and mixed primary cultures of adult human hepatocytes.

Specific polyclonal antibodies were used to investigate the distribution of two cytochrome P-450 isozymes (5 and 8), NADPH cytochrome c reductase, and epoxide hydrolase in adult human hepatocytes cultured alone or co-cultured with rat liver epithelial cells. The enzymes were localized by the indirect immunoperoxidase technique following fixation with a paraformaldehyde-glutaraldehyde mixture and membrane permeabilization with saponin. The pattern of distribution of the four enzymes after 24 hr in culture was similar to that found in vivo. Virtually all the hepatocytes exhibited nearly homogeneous positive staining for cytochrome P-450-8, whereas only 60-80% were positive for cytochrome P-450-5. Nearly homogeneous staining was also observed in all hepatocytes for NADPH cytochrome c reductase and epoxide hydrolase. During the first 12 days in pure culture, the intensity of staining, as well as the number of positively stained cells, decreased slightly except for epoxide hydrolase, which did not show any obvious change. In contrast, even after 15 days in co-culture the extent of staining for all the enzymes decreased less than in pure culture. These results indicate that adult human hepatocytes continue to express specific drug-metabolizing enzymes for several days in culture and provide further evidence that those cells are more stable than rodent hepatocytes in primary culture.

Adolescent↗