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. Hepatocyte cultures, Kupffer cell cultures or cocultures were treated with 0.1 ng/ml-10 microg/ml lipopolysaccharide (LPS). Within this concentration range, no significant toxicity was observed in either cell type. In LPS-exposed cocultures, tumor necrosis factor alpha (TNFalpha) levels rose up to 5 ng/ml within 5 h; nitric oxide (NO) levels increased up to 70 microM within 48 h of treatment, both in a dose-dependent fashion. The release of negative (albumin) and positive (alpha1-acid-glycoprotein) acute phase proteins from the hepatocytes was strongly down- and up-regulated, respectively. The simultaneous treatment of the cocultures with phenobarbital and LPS (10 ng/ml) or 3-methylcholanthrene and LPS (10 ng/ml) resulted in a strong down-regulation (85%) of the phenobarbital-induced cytochrome P450 (CYP) isoform CYP2B1 in the hepatocytes whereas the 3-methylcholanthrene-induced isoform CYP1A1 was only weakly affected (15%). This specific down-regulation of CYP2B1 was mediated exclusively by TNFalpha, released from the Kupffer cells. It was not linked with NO release from or inducible NO synthase activity in the hepatocytes. The TNFalpha release was not affected by the two xenobiotics. Acetaminophen tested in these cocultures showed no direct interaction with the Kupffer cells. The use of liver cell cocultures is therefore a useful approach to investigate the influence of intercellular communication on xenobiotic metabolism in liver.

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. The maximal induction of P450 isoenzymes CYP2B1/2B2 (20- to 25-fold) and CYP2C6 (6-fold) were found at 0.75 mM PB at both oxygen tensions. In contrast, the highest induction of CYP1A1/1A2 (3-fold), of CYP3A (2-fold), and EROD activity were found with 3 mM PB in 4% O2 cultures. CYP2B-dependent testosterone hydroxylation at positions 16 alpha/beta was elevated to a greater extent in 13% O2 cultures (96-fold at 0.75 mM PB) compared to 4% O2 cultures (42-fold). This activity was affected by the insulin concentrations and the insulin:glucagon ratio. With decreasing insulin concentration (100 to 1 nM) or with increasing insulin:glucagon ratios (1:100-1:0.1), the enzyme activity increased preferentially in 4% O2 cultures. The results of these investigations demonstrate that different tissue oxygen tension modulates the responsiveness of the cultured hepatocytes to the glucoregulatory hormones insulin and glucagon and this modulation results in a altered activity of cytochrome P450 isoforms.

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, 0.75 mm) or 3-methylcholanthrene (3-MC, 6.25 mum). On day 4, PB increased CYP2B1/B2 content 19-fold (4% O(2)) or 27-fold (13% O(2)), CYP2C6 six-fold at both oxygen tensions, and CYP3A2 two-three-fold only in 4% O(2) cultures. On day 9, CYP2B1/B2 was still significantly increased. A clear correlation was found between P-450 isoenzyme content and corresponding isoenzyme activities, as assayed by testosterone hydroxylation and EROD activity on day 4. 3-MC increased CYP1A1/2 11-fold and eight-fold in 4 and 13% O(2) cultures, respectively, and the EROD activity 37-fold (4% O(2)) and 30-fold (13% O(2)) on day 4. Activities were still measurable on day 9, whereas the corresponding apoproteins CYP1A1 was not detectable. These results suggest that hepatocytes are able to adapt their xenobiotic metabolism to different tissue oxygen tensions and confirm the likelihood that oxygen tension is an important modulator of the region-specific expression of xenobiotic metabolism in liver.

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: 0.1-3.3%) from 24-72 h after seeding. Induced alterations in ploidy, in the number of S-phase cells, the degree of binuclearity, and cellular protein content were determined by twin parameter protein/DNA flow cytometry analysis of intact cells and isolated nuclei. CPA and PB increased whereas DMSO decreased dose dependently the total number of S-phase cells. The changes differed within individual ploidy classes and were modulated by the oxygen tension. CPA increased and DMSO decreased the number of S-phase cells preferentially among the diploid hepatocytes at periportal-like oxygen tension. In contrast, PB increased binuclearity and S-phase cells mainly among the tetraploid hepatocytes at perivenous-like oxygen tension. Cellular protein content increased dose dependently after exposure to the hepatomitogens (CPA, PB) and decreased after exposure to DMSO at both oxygen tensions. Comparison with in vitro data proves that chemicals which interact with cells from the progenitor liver compartment (CPA, DMSO) exert their mitogenic activity best in cultures at periportal-like oxygen tension preferentially in diploid hepatocytes, whereas chemicals which affect cells from the functional compartment show a higher activity at perivenous-like oxygen tension. Physiological oxygen tension seems to be an effective modulator of the proliferative response of cultured rat hepatocytes similar to that expected for periportally or perivenously derived hepatocytes.

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 2.25 kGy) and 30% complementary feed or with a control diet. None of the parameters examined (food consumption, body weight, haematological analysis, histopathological inspection of thymus, lung, liver, spleen and kidney, DNA analysis of bone marrow cells and nuclei from liver cells by flow cytometry) showed any statistically significant association with the feeding regimen. Minor changes in ploidy of liver cells and cell cycling of bone marrow cells were detectable (wheat-irradiation dose-dependent increase in G2/M-phase bone marrow cells up to 0.6%, decrease of 8C nuclei up to 1.1% in liver cells). From the pattern of alterations observed in our study, radiolytic by-products of wheat irradiation with a spindle poison-like activity can be excluded. Polyploid cells do not accumulate within the 90-day feeding period. The minor effects on cell cycle and ploidy observed are qualitatively comparable with the effects seen after food restriction in animal studies. It is suggested that an altered composition of fatty acids (the components of wheat most sensitive to irradiation) is responsible for these marginal effects. Our findings may explain the earlier findings of Bhaskaram and Sadasivan (American Journal of Clinical Nutrition 1975, 28, 130-135) who reported an increase in the number of polyploid cells in the lymphocytes of malnourished children fed irradiated wheat. The most likely mechanisms for such an effect are adaptive, constitutively regulated processes, similar to those which respond to food restriction. It is concluded that the consumption of irradiated wheat does not, therefore, pose any health risk to humans.

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. The relative contributions of CYP1A1/2, CYP2B1/2, CYP2C6, CYP2C11, CYP3A and CYP4A isoenzymes were determined with ELISA using monoclonal antibodies raised against purified cytochromes P-450 from rat liver microsomes. The characterization of the CMF revealed that contaminations with mitochondria, nuclei and lysosomes are relatively low. Among these, membranes derived from the endoplasmic reticulum appeared to be the major organelle contaminant of the CMF. The matrix components laminin, fibronectin and collagen type IV were found in appreciable amounts. Hepatocytes from untreated rats, cultured for up to nine days on CMF/COL-coated plates, retained their relative cytochrome P-450 contents at 1.5-3-fold higher levels when compared to cells cultured on COL, fibronectin or laminin. Similarly, hepatocytes from phenobarbital-treated rats preserved the contents of barbiturate-inducible CYP2B1/2 and CYP3A proteins best when cultured on CMF/COL. After exposure of hepatocytes cultured on CMF/COL to phenobarbital from days 3-6, CYP3A proteins were enhanced more than twofold and CYP2B1/2, depending on the exposure level, increased 1.3-6-fold. After exposure to 3-methylcholanthrene, a threefold increase of CYP1A proteins was found in CMF/COL and laminin cultures. These results indicate that CMF/COL, as a substratum in rat hepatocyte cultures, regulates gene expression of cytochromes P-450 isoenzymes for up to 9 days and provides a matrix which enables the cells to respond qualitatively similar to the response observed in different zones of the liver. This activity cannot be replaced by single-matrix components.

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). The cells adhered firmly, exhibiting minimal spreading and remaining grouped in columns or in cell islands, and retained their liver-specific functions for more than 1 wk. Hepatocytes secreted substantially higher amounts of albumin than cells cultured on collagen-coated dishes, and on Days 1 and 9 in culture the total P-450 content was 72 and 40%, respectively, of that of freshly isolated cells. On Day 6, the 7-ethoxyresorufin-O-deethylase and the aldrin epoxidase activities were still more than 50% that of freshly isolated hepatocytes. Exposure to phenobarbital on Days 3 to 6 increased the total cytochrome P-450 content twofold; exposure to 3-methylcholanthrene increased the activity of the corresponding cytochrome P-450 isoforms to 20 times that observed in untreated cultures and 6 times that observed in freshly isolated cells. Thus, given the ease with which they are prepared, the use of crude membrane fractions combined with culture medium supplemented with aprotinin and selenium can facilitate the preparation of reproducible cultures suitable for long-term in vitro pharmacotoxicologic studies using rat hepatocytes.

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. Hepatocytes and isolated nuclei were analyzed by DNA-protein flow cytometry. An aggregate correction procedure was applied and the proportion of S-phase, diploid, tetraploid or octoploid hepatocytes as well as binucleated cells, were measured or calculated. The proportion of S-phase cells within the diploid hepatocytes increased with increasing concentration of TA up to 3.9-fold, whereas the corresponding increase in S-phase mononucleated tetraploid cells was only 1.8-fold. S-phase binucleate tetraploid cells showed no increase. In the tetraploid hepatocytes, the mitogenic stimuli was detectable only in cultures maintained at 4% O2. The relative contribution of binuclear cells was increased 1.5-fold in the octoploid cells. It is concluded that the mitogenic activity of TA initiates DNA synthesis in diploid hepatocytes in the G1 and in the following G2 cell-cycle phase, omitting karyogenesis. The cellular protein content is not affected which indicates that the mitogenic activity of the chemical is not necessarily associated with an increase in cellular protein content. The results obtained correspond well with data of in vivo studies. The method applied therefore allows the mitogenic activity of nongenotoxic carcinogens to be detected in vitro within 48 h and their mode of action to be elucidated.

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. When administered directly, all chemicals except 2-AAF induced a dose-dependent increase in gene-mutation frequencies. However, 2-AAF was mutagenic after oral administration. The highest inducible mutation frequencies found with the hepatocarcinogens were 8 to 10 times the spontaneous mutation frequency, but only approximately 10% of that found with the control mutagen ENU. The known enzymic activation capacity of the granulation tissue, and the mutagenic activity of other chemicals in this system already reported, suggest that after direct exposure, the prostaglandin H synthetase pathway or lipoxygenases are involved in the activation of the hepatotoxins to gene mutation inducing species in vivo. 2-AAF seems to be converted via stable intermediates to the ultimate mutagenic species in the extra-hepatic target tissues.

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. Variation in the exposure time and recovery period can affect cell cycle kinetics both qualitatively and quantitatively, whereas the distribution of DNA and protein content are affected quantitatively only. A 24-h exposure combined with a 24-h recovery period proved to be the most efficient approach. Each of the tested chemicals induced a specific, dose-dependent pattern of altered cellular DNA and protein content. Comparison with results obtained in other genotoxicity tests, and with data reported earlier, showed that this two-parameter protocol can be used to recognize and to characterize chemicals as clastogens, or as compounds with a combined cytostatic/clastogenic activity, or as spindle-poison-like compounds.

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. In vitro, AAI was 19 times more mutagenic than AAII at this low oxygen tension but exhibited only 4 times greater activity than AAII under standard culture conditions. These results indicate that the genotoxic activity of AA in mammals is mainly caused by AAI and that the exposure of cells to AAI and AAII in vitro at low pO2 corresponds more closely to the metabolic situation in vivo. Therefore, the mutagenic potency of the two chemicals can only be estimated correctly at tissue oxygen tension. The influence of pO2 on the mutation frequencies seems to arise from a modulation of the activation/detoxification pathways.

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. From DNA and protein histograms, alterations in cell-cycle kinetics, variations in the amount of DNA in individual G1-phase cells and the enhancement of or increased variation in the protein content of the exposed cells were determined. Each of the 5 chemicals induced a specific dose-dependent pattern of changes in the DNA and protein histograms. DNA dispersion was enhanced with VC, AM, BL and BE but not with PR. The cell cycle was blocked in the G2 phase with VC, at early S phase with amethopterin and, depending on the dose, at the G1 or G2 phase with bleomycin or at the S phase or G2 phase with benomyl. Practolol inhibited cells slightly in the S phase at the highest exposure level. Protein analysis allows cytotoxic activity (loss of proteins) or induced unbalanced growth (protein accumulation) of test compounds to be recognized. The results obtained imply that the proposed two-parameter DNA/protein analysis by flow cytometry is a suitable method for prospective testing of chemicals for their induction of structural or numerical chromosome aberrations. Simultaneously, a broad range of cytotoxic, cytostatic and cell-cycle perturbing activities of the test agents can be recognized.

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. After oral application of AA, a dose-dependent mutagenic activity was seen. In contrast a very weak and inconsistent mutagenic effect was seen after systemic application of MNNG. These observations suggest that after oral application AA is not detoxified efficiently and can exert its mutagenic activity in extrahepatic tissues whereas MNNG is detoxified to a large extent at the site of administration.

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. The estimated CD50 values (concentration at which 50% of the cells detach) were at least twice as large as the corresponding IC50 values for all 7 chemicals tested. For the toxic tributyltin salts the cell detachment assay was 30-60 times less sensitive than the cloning efficiency assay. However, both assays rank all compounds tested in the same sequence of toxicity as that known from in vivo studies.