Kupffer cell-mediated differential down-regulation of cytochrome P450 metabolism in rat hepatocytes.

Nonparenchymal cells, particularly Kupffer cells, might play an important role in the modulation of xenobiotic metabolism in liver and its pharmacological and toxicological consequences. This intercellular communication via the exchange of soluble factors was investigated in primary rat Kupffer cells and hepatocytes. Freshly isolated rat Kupffer cells were seeded onto cell culture inserts and cocultured with 5 day old serum-free rat hepatocyte monolayer cultures at a ratio of 1:1 for 2 days.

Effect of periportal- and centrilobular-equivalent oxygen tension on liver specific functions in long-term rat hepatocyte cultures.

The influence of periportal- and centrilobular-equivalent oxygen tensions on cellular functions and xenobiotic metabolism was investigated in rat hepatocytes cultured under 20% (air/CO(2)), 13% or 4% O(2) for up to 9 days on teflon membrane dishes, coated with crude membrane fractions and collagen. Protein content, total cytochrome P-450 content, 7-ethoxy-resorufin-O-deethylase (EROD) and the profile of hydroxytestosterone metabolites were not significantly affected by the oxygen tension, whereas intracellular lactate dehydrogenase activity and albumin secretion were increased at 4% O(2) in comparison with 13 and 20% O(2) cultures. The induction of P-450 isoenzymes and corresponding catalytic activity was determined on days 4 and 9, following a 3-day exposure to phenobarbital (PB, 0.

Oxygen tension, insulin, and glucagon affect the preservation and induction of cytochrome P450 isoforms in cultured rat hepatocytes.

The role of oxygen tension, insulin, and glucagon on the preservation and induction of cytochrome P450 isoenzyme activities and contents was investigated in rat hepatocytes cultured for 4 days on crude liver membrane fractions at 4 or 13% O2. At 13% O2, three out of six immunochemically analyzed P450 isoenzymes were significantly higher than in 4% O2. Exposure to phenobarbital (PB) from Days 1 to 4 dose dependently increased the protein content and decreased the albumin secretion in 13% O2 cultures only.

Physiological oxygen tension modulates the chemically induced mitogenic response of cultured rat hepatocytes.

Freshly isolated rat hepatocytes were cultured at periportal- (13% O2) or perivenous-like (4% O2) oxygen tension and exposed to subtoxic exposure levels of cyproterone acetate (CPA: 10-330 microM), phenobarbital (PB: 0.75-6 mM), and dimethylsulfoxide (DMSO: 0.1-3.

Cell-cycle and ploidy analysis in bone marrow and liver cells of rats after long-term consumption of irradiated wheat.

Rats were fed for 4 or 90 days either with 70% freshly irradiated wheat (0.25, 0.75 or 2.

Crude liver membrane fractions and extracellular matrix components as substrata regulate differentially the preservation and inducibility of cytochrome P-450 isoenzymes in cultured rat hepatocytes.

The influence of cell-substrata interactions on the preservation of basal or in-vivo-induced microsomal cytochrome P-450 isoenzyme contents in cultured rat hepatocytes and on the adaptive responses after exposure to phenobarbital or 3-methylcholanthrene in vitro, was investigated. Hepatocytes from untreated or phenobarbital-treated rats were cultured in serum-free, aprotinin-supplemented culture medium in 96-well microtiter plates coated with collagen type I (COL), laminin, fibronectin or crude liver membrane fractions/collagen type I (CMF/COL). Basal cell functions were characterized by measuring the total protein content and lactate dehydrogenase release.

Crude liver membrane fractions as substrate preserve liver-specific functions in long-term, serum-free rat hepatocyte cultures.

Over time, rat hepatocytes cultured on collagen lose the capacity to express liver-specific functions. The influence on this degradation process of an alternative substratum--crude membrane fractions prepared from the liver of the same rat strain--was investigated. Freshly isolated rat hepatocytes were cultured in serum-free Williams E medium supplemented with aprotinin, selenium, dexamethasone, and insulin in flasks coated with a mixture of rat liver crude membrane fractions:collagen type I (100:1).

Single cell analysis in toxicity testing: the mitogenic activity of thioacetamide in cultured rat hepatocytes analyzed by DNA/protein flow cytometry.

Rat hepatocytes were cultured at 4% O2 and 13% O2 and exposed to the nongenotoxic rodent carcinogen thioacetamide (TA) from 24 to 72 h after isolation at exposure levels between 0.01 and 0.33 mM.

Hepatocarcinogens induce gene mutations in rats in fibroblast-like cells from a subcutaneous granulation tissue.

Gene-mutations at the 6-thioguanine locus, in fibroblast like-cells rapidly proliferating on the inside of rat subcutaneous air pouches were analysed (Granuloma Pouch Assay). Target cells were exposed directly by injection into the air pouch, or systemically by oral or intraperitoneal administration to three hepatocarcinogens aflatoxin B1, 2-acetylaminofluorene (2-AAF) and vinylchloride. In addition ethylnitrosourea (ENU) was used as a positive control, and 2-aminofluorene to characterize the pharmacokinetic behaviour of 2-AAF.

Alterations in the cellular DNA and protein content determined by flow cytometry as indicators for chemically induced structural and numerical chromosome aberrations.

Cellular DNA and protein content were determined simultaneously in freshly isolated fibroblast-like rat cells by flow cytometry. After exposure to doxorubicin, nitrofurantoin, propranolol and practolol at a low, tissue like oxygen concentration (5% O2), drug-induced alterations in cell cycle kinetics, in the distribution of DNA and in the protein content of G1-phase cells (nucleus/cytoplasm ratio) were analysed. Optimal exposure time (5 or 24 h) and recovery interval (24 or 48 h) were determined.

Low oxygen tension, as found in tissues in vivo, alters the mutagenic activity of aristolochic acid I and II in primary fibroblast-like rat cells in vitro.

The mutagenic activities of aristolochic acid I (AAI) and II (AAII), the two main components of aristolochic acid (AA), were tested for mutagenicity in vivo in a subcutaneous granulation tissue in rats and in vitro in the corresponding freshly isolated and cultured target cells. In vivo at equimolar dose, AAI induced 16 times more 6-thioguanine resistant cells than AAII. Oxygen tension in vitro was adjusted to that found in vivo: in the subcutaneous connective tissue, the pO2 was determined to be 40 +/- 9 mm Hg, which corresponds in vitro to an O2 concentration of 5% in the incubator atmosphere.

A two-parameter flow cytometry protocol for the detection and characterization of the clastogenic, cytostatic and cytotoxic activities of chemicals.

Cultured, freshly-isolated rat fibroblasts were exposed in vitro to vincristine sulphate (VC), amethopterin (AM), bleomycin (BL), benomyl (BE) and practolol (PR). Cells treated for 5 h were subjected 24 h later to a two-parameter (DNA/protein) flow cytometry analysis. The fluorochromes used were sulphorhodamin 101 and DAPI.

Aristolochic acid induces 6-thioguanine-resistant mutants in an extrahepatic tissue in rats after oral application.

The mutagenic activity of the natural plant product aristolochic acid (AA) was tested in the Granuloma Pouch Assay, which detects gene mutations induced in a subcutaneous granuloma tissue of rats. After direct exposure of the target tissue, AA induced high frequencies of mutants at a relatively low cytostatic/cytotoxic level. AA was more potent that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at equimolar doses.

Cell detachment and cloning efficiency as parameters for cytotoxicity.

Cell detachment and cloning efficiency of Baby Hamster Kidney cells (BHK-21 C13) were used as parameters to quantify cytotoxicity in vitro of 3 endogenous chemicals (glutathione, L-methionine, L-cysteine HCl) and 4 organotin compounds (tributyltinoxide, tributyltinchloride, tetrabutyltin, tetraphenyltin). IC50 values (inhibitory concentration at which the cloning efficiency was reduced to 50%) were estimated to be larger than 10(-3) M for all 3 endogenous substances, which served as a calibration of the cell culture system for non-toxic chemicals. For the 2 tributyltin salts the IC50 values were estimated to be near 10(-6) M and for the 2 tetraalkyltin compounds near 10(-5) M.