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1.
Cell Biol Toxicol ; 7(4): 327-45, 1991 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1794108

RESUMO

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.


Assuntos
Expressão Gênica/genética , Genes ras , Fígado/citologia , Transfecção , 2-Acetilaminofluoreno/metabolismo , Albuminas/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Southern Blotting , Western Blotting , Divisão Celular , Linhagem Celular , Linhagem Celular Transformada , Células Cultivadas , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Fígado/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Transferrina/metabolismo
2.
Cell Biol Toxicol ; 5(3): 261-70, 1989 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2598085

RESUMO

The toxic effects of 7 beta-hydroxycholesterol (7 beta-OHC) on cultures and co-cultures of rat hepatocytes, rat liver epithelial cell lines, and rat liver fibroblast lines were investigated. Hepatocytes in primary culture or co-cultured with proliferative epithelial cells, were not affected by the presence of 7 beta-OHC at a concentration of 400 microM over a period of 72 hours. In contrast, proliferative cultures of liver epithelial cell lines and liver fibroblast lines were killed by 50 microM 7 beta-OHC within the first 24 hours. Established liver epithelial cells (hyperploid) were more sensitive to 7 beta-OHC than the same line at early passages (diploid). When hepatocytes and liver epithelial cells were co-cultured and treated with 100 microM 7 beta-OHC, only epithelial cells were lysed. A concentration of 50 microM 7 beta-OHC was toxic to co-cultures of liver epithelial cell and fibroblasts together. In a serum-free medium, the cytotoxic concentration of 7 beta-OHC was lower than that in the serum-supplemented medium. Thus, liver epithelial cells cultured alone or co-cultured with hepatocytes were killed at 12.5 microM and 50 microM 7 beta-OHC, respectively. Finally, cholesterol concentrations four-fold that of 7 beta-OHC antagonized the lethal effects of 7 beta-OHC in the serum-free medium.


Assuntos
Hidroxicolesteróis/toxicidade , Fígado/efeitos dos fármacos , Albuminas/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colesterol/toxicidade , Complemento C3/metabolismo , Células Epiteliais , Fibroblastos , Fígado/citologia , Fígado/metabolismo , Ratos , Ratos Endogâmicos , Transferrina/metabolismo
3.
J Chromatogr ; 488(1): 237-48, 1989 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-2715283

RESUMO

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.


Assuntos
2-Acetilaminofluoreno/metabolismo , Cocarcinogênese , Fígado/metabolismo , 2-Acetilaminofluoreno/toxicidade , Animais , Biotransformação , Células Cultivadas , Cromatografia Gasosa , Epitélio/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Oxirredução , Ratos
4.
Cell Biol Toxicol ; 3(3): 327-40, 1987 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3507261

RESUMO

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.


Assuntos
2-Acetilaminofluoreno/metabolismo , Fígado/metabolismo , Compostos de Organossilício , Silício/metabolismo , 2-Acetilaminofluoreno/análise , Animais , Radioisótopos de Carbono , Cromatografia Gasosa-Espectrometria de Massas , Ratos , Silício/análise
5.
C R Seances Soc Biol Fil ; 180(4): 434-40, 1986.
Artigo em Francês | MEDLINE | ID: mdl-2948623

RESUMO

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.


Assuntos
2-Acetilaminofluoreno/metabolismo , Fígado/metabolismo , Animais , Cromatografia Gasosa , Técnicas In Vitro , Espectrometria de Massas , Ratos , Ratos Endogâmicos
6.
C R Seances Soc Biol Fil ; 180(4): 460-72, 1986.
Artigo em Francês | MEDLINE | ID: mdl-2948626

RESUMO

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.


Assuntos
Meios de Cultura , Fígado/citologia , Animais , Linhagem Celular , Células Epiteliais , Masculino , Ratos , Ratos Endogâmicos , Fatores de Tempo
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