Metformin induces an agonist-specific increase in albumin production by primary cultured rat hepatocytes.

Metformin (MET) is known to increase several biological effects of insulin (INS), but there is no information concerning its direct effects on protein synthesis. We studied the action of MET on albumin production by primary cultures of freshly isolated rat hepatocytes, alone or in combination with various agonists: INS, IGF-1, EGF, thyroxin, and dexamethasone. While having no effect alone, MET in vitro potentiates the effects of INS, IGF-1, and EGF. When this increasing effect toward INS was studied over a broad concentration range, MET appeared to improve low-acting INS levels and to intensify the maximal INS effects. In contrast, MET did not change the production of albumin stimulated by thyroxin or dexamethasone. Animals chronically pretreated with MET in vivo showed a higher yield of isolated hepatocytes, better attachment, and especially higher viability after liver perfusion and during cell culture. This may largely explain why basal albumin rates were higher than in in vitro-treated cells. The effect of MET in the presence of the agonists exhibited the same agonist-specificity as in vitro. Our data provide new insights into the pharmacology of MET by showing that hepatic protein synthesis is increased by MET and INS. From the specificity of action of MET towards INS, IGF-1, and EGF (but not thyroxin or dexamethasone), we hypothesize that this biguanide may act on intracellular pathways located between membrane receptors and sites of branching in the signaling cascades shared by these agonists.

[Immortalization of cultured cells by the Ha-rasEJ oncogene and expression of a gene of biogenetic value]. / Immortalisation de cellules en culture par l'oncogène Ha-rasEJ et expression d'un gène d'intérêt biogénétique.

The double recombination into NIH-3T3 cells of the cloned Ha-rasEJ oncogene and the cDNA gene of the human growth hormone (hGH) under the control of a heat inducible promoter (hsp70) allowed hGH production either in vitro using the mass culture of engineered cells in biogenerators, or in vivo after xeno-transplantation of the cells into an animal host. Therefore the in vivo synthesized hGH induced the production of anti-hGH polyclonal antibodies.

Maintenance of liver function in long term culture of hepatocytes following in vitro or in vivo Ha-rasEJ transfection.

Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-rasEJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 micrograms albumin and 0.32 microgram transferrin per 10(6) cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminofluorene (2-AAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 micrograms albumin and 11.0 micrograms transferrin per 10(6) cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGH per 10(6) cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 micrograms albumin and 11.7 micrograms transferrin per 10(6) cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AAF per mg cell protein. Hence, Ha-rasEJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.

Transfection of lipoma cells with papilloma bovine virus subgenomic fragment.

Lipoma cells with consistent chromosomal aberration have been transfected with plasmids carrying papilloma bovine virus subgenomic fragment (PBV 69). The successful transformation of the cells was ascerted on the changed growth pattern of the cells in liquid medium, colony formation in soft agar and modified cell appearance in electron microscopy; transfection with PBV 69 has not been, however, sufficient to immortalize lipoma cells.

Gas chromatographic-mass spectrometric study of deacetylation and oxidation of 2-acetylaminofluorene by rat liver epithelial cell lines upon cocarcinogen induction.

Cell line cultures from postnatal and adult rats were incubated with 5-100 mumol/l [9-14C]-2-acetylaminofluorene. On incubation of 10 mumol/l, ring-hydroxylated metabolites, expressed as nmol hydroxy-2-acetylaminofluorene (OH-2-AAF)/mg cell protein/24 h, were 9-OH- 1.28 +/- 0.37, 7-OH- 1.08 +/- 0.28 and 5-OH- 0.30 +/- 0.08, and deacetylated 2-AAF (2-AF) 1.20 +/- 0.18. For 5, 10, 50 and 100 mumol/l 2-AAF, the total production of OH-2-AAF (same units) and 2-AF (%) were, respectively, 0.86 (0%), 3.86 (35%), 17.8 (60%) and 35.03 (89%). On preincubation with phenobarbital (BP) or 3-methylcholanthrene (3-MC) and then incubation of 10 mumol/l 2-AAF, the total synthesis of OH-2-AAF increased 1.9-fold (PB) and 2.5-fold (3-MC). In addition, four other OH-2-AAF (1-OH-, 3-OH- and two unknown OH-2-AAF) were produced and glucuronidation of all metabolites was induced and amounted to 57% of the total after PB and 75% after 3-MC preincubation. Metyrapone or alpha-naphthoflavone inhibition of BP or 3-MC, respectively, markedly affected the production of free and conjugated metabolites and, almost completely, the deacetylation of 2-AAF.

Gas chromatography-mass spectrometry of perindopril and its active free metabolite, an angiotensin convertase inhibitor: choice of derivatives and ionization modes.

Perindopril, a perhydroindole compound and a novel class of angiotensin convertase inhibitor, after oral administration leads to an active metabolite by de-esterification of an ethyl ester. Routine biological measurements are currently done using a radioimmunological assay, but a mass fragmentographic method was developed using plasma spiked with the drugs, which were then derivatized to the isobutyl ester heptofluorobutyramide and assayed using ammonia negative chemical ionization. Levels of 100 pg/ml were assayed. However, isobutanol derivatization provoked partial transesterification of the ethyl ester of the parent drug into the diisobutyl ester derivative, which corresponds to the active metabolite. A second method of derivatization to stable trimethylsilyl esters preserved the original ethyl ester of the parent drug. Despite the lower ionization yields, the mass fragmentographic method was sensitive and accurate enough to work satisfactorily at the 2 ng/ml level in spiked plasma, which is the level found currently in patients.

Gas chromatographic-mass spectrometric assessment of the pharmacokinetics of amineptine and its main metabolite in volunteers with liver impairment.

A pharmacokinetic study of amineptine (Survector) and its C5 metabolite, resulting from a beta-oxidation of the heptanoic acid side-chain, was undertaken with ten human volunteers, who received a single 100-mg tablet of amineptine orally. They were affected with liver impairment in order to determine if this situation would alter greatly the pharmacokinetic parameters. The internal standard was the octanoic acid homologue. Analyses were carried out by gas chromatography (GC) and GC-mass spectrometry using TMS ester derivatives. Plasma samples were extracted using a C18 reversed-phase cartridge at pH 4.0. Mass fragmentographic measurements on the plasma samples were performed on the m/z ions (M + H)+ and (base peak)+ using ammonia chemical ionization. The global evaluation of precision was good and the coherence between the two modes of measurements, (base peak)+ and (M + H)+ ions, gave a regression factor r close to unity. For amineptine the total body clearance and mean residence time were accurate and precise with eight volunteers, but only four volunteers showed such coherent data for the slope of the elimination curve, beta, and half-life. However, the beta value, half-life and mean residence time of the C5 metabolite were accurate and precise with seven, eight and ten volunteers, respectively. It is concluded that the drug was still detoxified at normal levels.

Synthesis and gas chromatographic-mass spectrometric analysis of reduced compounds derived from 6 alpha- and 6 beta-hydroxy-11-deoxycorticosterone.

A series of thirty two 6-hydroxylated steroids were synthesized by selective reduction of the 4-5 double bond, the 3-oxo group, and/or the 20-oxo group of 6 alpha- and 6 beta-hydroxyDOC. The different reactions leading to the production of specific isomers are discussed. The gas chromatographic and spectrometric characteristics of the methoxime-trimethylsilyl (MO-TMS) or trimethylsilyl (TMS) derivatives of the isomers obtained are given. The gas chromatographic separation of the syn- and anti-isomers of the methoxime in position 3 was found to be characteristic of the configuration of the hydroxyl in position 6. The difference between methylene unit values of syn- and anti- isomers is much larger for the 6 alpha-series than for the 6 beta-series. The mass spectral analysis showed that many ions are specific of the MO-TMS derivatives of steroids with 3,6-dihydroxy-4-ene or 3-oxo-6-hydroxy-4-ene structure. In the case of steroids with a saturated ring A no significant ions characteristic of the presence of a 6-trimethylsilyloxy substituent were found. This work provides previously unavailable reference data on 6-hydroxylated steroids which should facilitate the study of corticosteroid metabolism.

A serum-free medium with or without a carrier protein supports the growth of the Y-1 mouse adrenocortical cell line and its steroidogenic function.

The development of a method for the serum-free culture of the Y-1 mouse adrenocortical tumor cell line has permitted a detailed search for factors regulating cellular growth and steroidogenesis. The serum-free medium (SFM) was made of Ham's F10 basal medium supplemented with free fatty acids adsorbed on albumin. The SFM complemented with calcium, arachidonic acid and cholesterol, i.e. SFM-S, induced cell proliferation to a density at confluency higher than that obtained with 1% serum-supplemented medium (1%-SSM) and allowed to sustain cell growth for more than six passages. When albumin was replaced by a dextran polymer (Mr = 2 x 10(6)) used as a carrier of lipids instead of albumin (which resulted in a serum-free and protein-free medium, SPFM), the cell number was 75% of that observed with the SFM-S. The addition to SPFM of beta-globulin alone or combined with insulin caused a 2- or 3-fold increase in the final cell density, respectively. The ability of the Y-1 cell line to produce steroids in response to ACTH was found to be higher in SFM-S or SPFM than in 1%-SSM. Furthermore, the addition of beta-globulin to SPFM stimulated steroid hormone biosynthesis with a marked increase in 11 beta-hydroxylated steroid production. These studies demonstrate that the use of a defined mixture of nutriments and of few growth factors permits to sustain not only the cellular proliferation of the Y-1 cell line but also its differentiated function of ACTH-induced steroidogenesis.

Gas chromatography-mass spectrometry analysis of tert.-butyldimethylsilyl derivatives of 2-acetylaminofluorene and metabolites in isolated rat hepatocytes.

A new technique for the conversion of 2-acetylaminofluorene and several ring-hydroxylated metabolites to mono- and di-tert.-butyldimethylsilyl derivatives was developed to permit their analysis by gas chromatography-mass spectrometry in order to quantify the metabolism of 2-acetylaminofluorene incubated in freshly isolated rat hepatocytes. This new gas chromatography-mass spectrometry method allowed the separation, identification and quantitation of seven known metabolites comprising five arylhydroxylated compounds, 2-aminofluorene and N-hydroxy-2-acetylaminofluorene.

Gas chromatography-mass spectrometry of isobutyl ester trimethylsilyl ether derivatives of bile acids and application to the study of bile sterol and bile acid biosynthesis in rat liver epithelial cell lines.

The derivatization of bile acids into trimethylsilyl ether isobutyl ester (IBTMS) and of neutral sterols into trimethylsilyl ether (TMS) allowed the separation on an OV-1 capillary gas chromatography column of 15 bile steroids as follows: cholesterol, 7 alpha-hydroxycholesterol, 6 beta-hydroxycholesterol, 6 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, lithocholate, deoxycholate, 25-hydroxycholesterol, chenodeoxycholate, cholate, murocholate, hyodeoxycholate, ursodeoxycholate, hyocholate, and beta-muricholate. Fragmentation data of the coupled gas chromatographic-mass spectrometric (GC-MS) analysis of these nine bile acids as IBTMS derivatives under electron impact and chemical ionizations (methane, isobutane, and ammonia) are given. The ammonia chemical ionization appears to be the best mode for compound identification and quantitation due to fragmentations into high mass ions. The comparison of methylene units of the five sterols as TMS derivatives and of each type of methyl, TMS, or isobutyl ester of the nine bile acids as TMS ethers showed that isobutyl esterification increased dramatically the retention time of the bile acids, allowing their separation after the neutral sterols. Different methods of GC-MS analysis were applied to the study of bile steroid secretion in long-term rat liver epithelial cell lines, either serum-supplemented cell lines or serum-free cell lines, growing in serum-free medium since the primary explanation or after adaptation of serum-supplemented lines to this medium. It is demonstrated for the first time that liver epithelial cell lines maintain the metabolic pathway leading from synthesized cholesterol to dioxygenated sterols and the two normal main primary bile acids of the liver, chenodeoxycholic acid and cholic acid, up to 32-47% of the in vivo daily rate, and in addition the production of alpha-muricholic acid, the bile acid marker of murine liver.

[Biosynthesis of metabolites of 2-acetylaminofluorene by isolated hepatocytes of rats: study by gas chromatography-mass spectrometry]. / Biosynthèse des métabolites du 2-acétylaminofluorène par des hépatocytes isolés de rat: étude par chromatographie gazeuse-spectrométrie de masse.

A new technique for the derivatization, the separation and the quantification of 2-acetylaminofluorene (2-AAF) and its metabolites biosynthetized by freshly isolated hepatocytes was developed combining gas chromatography and mass spectrometry. Analysis of the different metabolites was carried out after their derivatization into tertbutyldimethylsilyl compounds. Freshly isolated hepatocytes metabolized 2-AAF and produced five aryl-hydroxylated compounds as well as the N-hydroxy-2AAF and the 2-aminofluorene. The metabolites were found under their free and conjugated forms.

[Culture of epithelial cell lines in chemically defined media on microcarriers in biogenerators]. / Culture de lignées de cellules épithéliales dans des milieux chimiquement définis sur microsupports en biogénérateurs.

A rat liver epithelial cell line has been propagated on microcarriers in 11 or 21 laboratory culture vessels for cell culture in suspension on microcarriers (biogenerators) with Ham F10 or DME as basal synthetic culture medium either serum-supplemented (SSM), or serum-free (SFM), or serum- and protein-free (SPFM). Without serum, the use of DME allows a cellular growth in the biogenerator at least equivalent to that obtained in culture dishes. For the cultivation on microcarriers in SFM in a biogenerator the use during the first day of culture of spent serum-free medium previously incubated (SFMI) in confluent culture dishes avoids the substratum treatment with serum. Results concerning the Vero cell line cultured in SPFM are shown.

The cytotoxicity of bleomycin A2 on adult rat liver epithelial cell lines at different stages of spontaneous cell transformation.

Cytotoxic effects of Bleomycin A2 on adult rat liver epithelial cell lines were evaluated by three methods: the incorporation rate of (3H) thymidine for DNA biosynthesis, the incorporation rate of L-(3H) leucine for protein biosynthesis and Giemsa dye staining of surviving cells. Chromosome investigations at successive passages of cell lines have shown that spontaneous chromosome abnormalities in distribution and structure after 15-20 passages, i.e. 50 to 60 cell generations, were the earliest morphological sign of spontaneous transformation. In this study a highly spontaneously transformed cell line was very sensitive to the drug. Another cell line at the beginning of spontaneous transformation appeared to be insensitive although on further passage it became more sensitive. The use of microtitration plates made it easier for us to undertake a comparative study of the different parameters. Following Bleomycin A2 exposure, Giemsa staining gave the best evaluation of cell killing whereas thymidine incorporation allowed the estimation of cell recovery. The antineoplastic effect of Bleomycin A2 can probably be used to evaluate the malignant potential of different rat liver epithelial cell lines.

Biosynthesis of sterols and bile acids in rat liver epithelial cell lines.

A rat liver epithelial cell line growing in a serum-supplemented medium expressed biosynthetic pathways of bile sterols and of free and conjugated chenodeoxycholic and cholic acids, the main primary bile acids of the liver. They were identified and measured by gas chromatography-mass spectrometry. The bile steroid secretion in the serum-supplemented cell line was established upon incubation in a serum-free medium which was demonstrated to sustain cell growth, allowing elimination of the interference of exogenous bile steroids and effectors. The free bile acid secretion was also expressed in a subline adapted to proliferate in this serum-free medium, i.e., a basal medium supplemented with 4 g/l albumin carrying 7.6 muequiv./l of a mixture of six long-chain free fatty acids but without any addition of hormones and growth factors. In addition, the rat liver epithelial cell line growing in the serum-supplemented medium maintained, with time, a steady-state of bile acid secretion over a lifespan of 500 days. In the two types of liver epithelial cell lines, dexamethasone and chenodeoxycholic acid supplementation exerted, individually, either a stimulating or an inhibiting effect on the bile acid secretion concurrently with the hydroxylation of chenodeoxycholic acid into alpha-muricholic acid.

Rat liver epithelial cell cultures in a serum-free medium: primary cultures and derived cell lines expressing differentiated functions.

Rat liver epithelial cells explanted in a serum-free medium (SFM) composed of Ham's F10 basal medium plus free fatty acids adsorbed on bovine albumin gave successful rise to primary cultures and then to long-term cell lines that expressed liver functions; induction of L-tyrosine aminotransferase by glucocorticoids, hepatic pattern of progesterone metabolism, and biosynthesis of murine primary bile acids; chenodeoxycholic and cholic acid common to higher vertebrates and alpha-muricholic acid specific of the rat bile.

Time course study of L-tyrosine aminotransferase induction in rat liver cell lines.

The enhancement of L-tyrosine aminotransferase activity by dexamethasone, an exclusive function of the liver, was serially measured at different passages of eight rat liver epithelial cell lines initiated and continuously grown in either a serum-supplemented medium or a serum-free medium. The enzyme basal activity was found to be 5.4 +/- 1.8 mU for cell lines in serum and 6.8 +/- 3.4 mU for cell lines without serum. Under the influence of dexamethasone (10(-6) mol/l for 5 hours) this basal level could be increased up to 2.9 fold in the presence of serum and 2.5 fold in its absence when investigations were carried out at early passages. During the following subcultures the induction ratio gradually declined and scarcely any induction could be detected after the 15th passage for cells grown in serum and after the 25th passage for cell lines grown without serum.

Oxidoreductive and hydroxylating metabolism of progesterone in rat liver epithelial cell lines.

Mechanisms and sequences of reduction and hydroxylation of progesterone into 6-hydroxypregnanolones were studied in proliferating rat liver epithelial cell cultures. These cell lines had an intense metabolic activity and all the metabolites were unconjugated. The formation of 3 alpha,6 alpha- and 3 beta,6 alpha-dihydroxy-5 alpha-pregnan-20-one was observed when the cells were incubated with progesterone, 5 alpha-pregnane-3,20-dione, 3 alpha- or 3 beta-hydroxy-5 alpha-pregnan-20-one and 6 alpha-hydroxy-5 alpha-pregnane-3,20-dione but not with 6 alpha- or 6 beta-hydroxyprogesterone, 5 beta-pregnane-3,20-dione, 3 alpha- or 3 beta-hydroxy-5 beta-pregnan-20-one. These findings indicate that the potential precursors of the 6 alpha-hydroxypregnanolones have a 5 alpha-configuration. The reduction of 5 alpha-pregnane-3,20-dione at C-3 followed by a 6 alpha-hydroxylation can be postulated as the major, if not the only, metabolic pathway. However, the possibility that 6 alpha-hydroxylation may occur prior to reduction of the C-3 oxo group cannot be entirely ruled out. The stereospecificity of reduction at C-5 and hydroxylation at C-6 are discussed and compared with 6-hydroxylated progesterone metabolites found in man and some other mammals during pregnancy and the neonatal period.

Interactions of caffeine and phenobarbital on sister chromatid exchanges in rat liver epithelial cell lines treated with 2-acetyl-aminofluorene and N-acetoxy-2-acetylaminofluorene.

Using "Fluorescence plus Giemsa" technique, sister chromatid exchanges (SCE) were determined on second-division metaphases in rat liver epithelial cell lines treated with 2-acetylaminofluorene (2-AAF), the procarcinogen, and N-acetoxy-2-AAF (N-OAc-2AAF), an ultimate carcinogen analog. The SCE frequency was found decreased after some conditions of 2-AAF treatment and increased with N-OAc-2-AAF. Phenobarbital (PB) decreased also the SCE frequency but cancelled the 2-AAF action when incubated after the procarcinogen. The addition of caffeine (Caf) cancelled the action of 2-AAF but not of phenobarbital. It is suggested that the mechanism of SCE may have several origins.

Laser nephelometric measurement of seven serum proteins compared with radial immunodiffusion.

Quantification of immunoglobulins IgG, IgA, IgM, C3 complement, siderophilin, alpha 2-macroglobulin and haptoglobin in serum by a Behring laser nephelometer coupled with a date processing apparatus was compared with values obtained by the radial immunodiffusion method of Mancini. The precision was not improved by the use of the laser nephelometer as compared to Mancini radial immunodiffusion, but within run precision was better than that among runs for both methods. The analytical recovery studies for laser nephelometry showed a close concordance between the theoretical and measured values for all proteins. The two techniques were also compared by means of correlation studies.