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P Maier

Publications and source records attributed to P Maier.

At least 37 records · Page 2Linked to original sources
PubMed1995

Nuclear matrix condensation and c-myc and c-fos expression are specifically altered in culture rat hepatocytes after exposure to cyproterone acetate and phenobarbital.

Regenerative or hyperplastic growth promotes carcinogenesis and can be induced by many nongenotoxic carcinogens. The mitogenic potential of the rodent liver tumor promoters, cyproterone acetate and phenobarbital was investigated in primary rat hepatocyte cultures. Two premitotic markers were analyzed, the expression of two immediate-early genes (c-fos and c-myc) and the decrease in the nuclear quinacrine dihydrochloride fluorescence indicative for a G0-G1 cell cycle shift. C-fos expression and decrease in nuclear fluorescence could be induced by both chemicals, phenobarbital being the lesser potent, whereas c-myc expression was only inducible by cyproterone acetate. In situ hybridization with c-myc revealed that both chemicals enhanced c-myc mRNA levels in individual cells, however the number of responding hepatocytes was increased by cyproterone acetate only. The chemical-induced premitotic changes in hepatocytes were highly specific in terms of affected genes and ploidy levels of responding hepatocytes.

Animals↗
PubMed1995

Hepatocyte-derived interleukin-6 and tumor-necrosis factor alpha mediate the lipopolysaccharide-induced acute-phase response and nitric oxide release by cultured rat hepatocytes.

The regulation of acute-phase protein production and nitric oxide (NO) release in lipopolysaccharide-induced liver injury is thought to occur in response to monocytes/macrophages and Kupffer-cell-derived cytokines. In this study, we used primary cultured rat hepatocytes maintained as a differentiated phenotype to investigate the direct effects of endotoxin (lipopolysaccharide) on the production of the acute-phase proteins and on NO release. Lipopolysaccharide (10 micrograms/ml) increased the production of alpha 2-macroglobulin 2.5-fold compared to untreated cultures and decreased the production of albumin by 50%. The effect of lipopolysaccharide was mimicked by adding interleukin-6 (IL-6) and tumor-necrosis factor alpha (TNF-alpha), cytokines being induced by treatment of hepatocytes with lipopolysaccharide. Maximal TNF-alpha (600 pg/ml) and IL-6 (1800 pg/ml) concentrations were observed 4 h and 6 h after lipopolysaccharide stimulation, respectively. The lipopolysaccharide-induced acute-phase protein response was blocked by anti-(IL-6) but not by anti-(TNF-alpha) IgG. The latter reduced the lipopolysaccharide-induced IL-6 production by 60%. Besides its effects on the acute-phase proteins, endotoxin caused a significant increase in NO production in cultured rat hepatocytes. Unlike anti-(IL-6) IgG, anti-(TNF-alpha) IgG reduced the lipopolysaccharide-induced NO production by 50% indicating that endotoxin-induced NO production is partially mediated by TNF-alpha but not by IL-6. Preculture with gadolinium chloride (GdCl3), an inhibitor of Kupffer cells, did not change the response of hepatocytes to lipopolysaccharide indicating that the observed findings are direct endotoxin effects on hepatocytes. The data demonstrate that by their production of TNF-alpha and IL-6 rat hepatocytes respond to lipopolysaccharide treatment with an IL-6 mediated acute-phase protein and a TNF-alpha-mediated NO production. These features have previously been attributed to monocytes/macrophages and Kupffer cells.

Acute-Phase Reaction↗
PubMed1995

Tumor necrosis factor alpha differentially modulates the cellular response of rat hepatocytes in periportal- and pericentral-equivalent cultures.

Alterations of cellular functions induced by recombinant human tumor necrosis factor alpha (TNF alpha) were compared in rat hepatocytes cultured under either periportal-equivalent (10 nM insulin; 10 nM glucagon; 13% O2) or perivenous-equivalent conditions (10 nM insulin; 1 nM glucagon; 4% O2). TNF alpha induced a time- and dose-dependent increase in nitric oxide (NO) production and an acute phase response (inhibition of albumin secretion and elevation of alpha 2-macroglobulin production) under both culture conditions. NO production was more pronounced in periportal cultures, while the acute phase response was stronger in pericentral cultures. This suggests that NO production and the acute phase response are controlled by different pathways. After exposure to TNF alpha, DNA content was measured fluorimetrically and biochemically. A marked decrease in nuclear DNA content was found exclusively in pericentral cultures after an 8-h exposure, followed by an elevation of lactic dehydrogenase (LDH) release after a 12-h exposure. Aurintricarboxylic acid (100 microM), an inhibitor of endonuclease, significantly inhibited the TNF alpha-induced decrease in nuclear DNA content but only partially inhibited the LDH release. This indicates that the loss of nuclear DNA content in pericentral cultures is due to an activation of endonuclease and the resulting DNA fragmentation and does not correlate with NO production. Furthermore, the release of LDH seems to be only partially associated with DNA damage. Dexamethasone (100 nM) completely inhibited both TNF alpha-induced DNA fragmentation and the elevation of LDH release. The results clearly indicate that the toxicity of TNF alpha is influenced by the metabolic state of hepatocytes. Accordingly, the preferential perivenous cell injury observed after exposure to endotoxins in vivo seems to be due to a higher sensitivity of the pericentrally localized hepatocytes towards TNF alpha rather than a TNF alpha concentration gradient.

Animals↗
PubMed1995

Transforming growth factor beta 1 preferentially induces apoptotic cell death in rat hepatocytes cultured under pericentral-equivalent conditions.

The cytotoxicity of transforming growth factor beta 1 (TGF beta 1) was assessed in rat hepatocytes cultured under periportal (PP)-or pericentral (PC)-equivalent conditions. TGF beta 1 induced a 5-fold greater DNA fragmentation and LDH release in PC cultures than in PP cultures. At low exposure level (1 ng/ml TGF beta 1), albumin secretion and mitochondrial activity (rhodamine-123 uptake) were selectively reduced in PP cultures, whereas the incidence of apoptotic cells in PC cultures was about 10-fold higher than that in PP cultures. The time profiles of TGF beta 1-induced apoptotic and necrotic events and the concentration-response relationship differed in PC and PP cultures. In PC cultures the early appearance of cells with apoptotic nuclei was not associated with DNA fragmentation nor with an increase in LDH release or impaired mitochondrial function. At a high exposure level (5 ng/ml TGF beta 1), again cells with apoptotic nuclei were much more strongly induced in PC cultures but DNA fragmentation, LDH release, and impairment of mitochondrial activity all increased in an exposure-time dependent manner in both PP and PC cultures. At this exposure level 48 and 72% of the apoptotic cells detected in PC cultures after continuous exposure for 24 hr were induced within an exposure of 1 and 4 hr, respectively. Aurintricarboxylic acid (50 microM), an inhibitor of endonucleases, significantly inhibited the appearance of apoptotic cells and the progression in apoptosis. Clearly, TGF beta 1 preferentially induced apoptotic cell death in hepatocytes with PC-equivalent metabolism at low exposure levels. High exposure levels or prolonged exposure periods produced both apoptosis and necrosis.

Animals↗
PubMed1995

Growth hormone-releasing peptide-6 stimulates sleep, growth hormone, ACTH and cortisol release in normal man.

The synthetic hexapeptide growth hormone-releasing peptide (GHRP-6) stimulates growth hormone (GH) release in animals and man. GH-releasing hormone (GHRH) has the same effect. In addition, pulsatile administration of GHRH in normal men results in increased slow-wave sleep (SWS) and blunted cortisol levels. The effect of GHRP on nocturnal hormone secretion and on the sleep electroencephalogram (EEG) is still unknown. We compared the effect of repetitive i.v. boluses (4 x 50 micrograms) of GHRP and placebo (PL) on the sleep EEG (23.00 to 07.00 h) and on the secretion profiles of GH, ACTH and cortisol (20.00 to 07.00 h) in normal male controls. After GHRP, the GH concentration (22.00 to 03.00 h) increased (15.4 +/- 9.6 ng/ml after GHRP vs. 5.5 +/- 4.0 ng/ml after PL, p < 0.02), as did the ACTH level (22.00 to 02.00 h: 21.0 +/- 5.3 pg/ml after GHRP vs. 16.6 +/- 3.1 pg/ml after PL, p < 0.02). During the total night, and particularly during the first half of the night, cortisol secretion was enhanced (22.00 to 03.00 h: 56.0 +/- 31.0 ng/ml after GHRP vs. 25.2 +/- 9.0 ng/ml after PL, p < 0.02). Stage 2 sleep increased (270.1 +/- 25.3 min after GHRP vs. 245.4 +/- 25.8 min after PL, p < 0.02), whereas other sleep-EEG variables including SWS remained unchanged. Our data demonstrate that GHRP stimulates not only GH release but also hypothalamic-pituitary-adrenocortical hormone secretion. The latter effect is opposite to the blunting of cortisol after GHRH. Both GHRP and GHRH promote sleep. However, GHRP enhances stage 2 sleep and does not affect SWS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1994

Cultured rat hepatocytes adapt their cellular glycolytic activity and adenylate energy status to tissue oxygen tension: influences of extracellular matrix components, insulin and glucagon.

The influence of extracellular matrix components, insulin, and glucagon on the cellular response to periportal- or pericentral-equivalent tissue oxygen tension was investigated in freshly isolated rat hepatocytes cultured at 13% O2 or 4% O2 in Teflon membrane dishes. With extended culture time, significant increases in lactate release and cellular lactate content were observed in cultures at 4% O2 compared with 13% O2. This shift toward glycolysis was detectable when hepatocytes were cultured on dishes coated with rat liver crude membrane fraction (CMF/COL) but not in collagen type I-coated dishes. This indicates that extracellular matrix components are involved in the process of adaptation. ATP and total adenylate content in cells cultured at 4% O2 were up to 40% lower than in cells cultured at 13% O2. However, the adenylate energy charge was not affected, suggesting that an adequate energy supply was maintained also in hepatocytes cultured at pericentral-equivalent oxygen tension. This adaptation was reversible. When hepatocytes were transferred either from 4% to 13% O2 or from 13% to 4% O2, they adapted the corresponding metabolic profile to the new oxygen tension within 2 days. This demonstrates that hepatocytes are not fully unidirectionally programmed. The modulation of the glycolytic activity by insulin and glucagon was effective in cultures at pericentral-equivalent oxygen tension (4% O2) only. Insulin (0.1-100 nM) shifted cellular metabolism toward the glycolytic pathway and glucagon (1-100 nM) counteracted the effect of insulin in a dose-dependent manner. Clearly, oxygen tension is the principal regulator in the hepatic glycolytic activity, whereas the hormones (insulin and glucagon) act as secondary modulators.

Adenosine Triphosphate↗
PubMed1994

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.

Albumins↗
PubMed1994

Nongenotoxic carcinogens shift cultured rat hepatocytes into G1 cell cycle phase: influence of tissue oxygen tension on cells with different ploidy.

The transition of quiescent freshly isolated rat hepatocytes (G0 phase) into a prereplicative stage of the cell cycle (G1 phase) has been visualized by a decrease in fluorescence of quinacrine dihydrochloride (QDH)-stained nuclei. This transition might be used as an early detectable and sensitive marker for the identification of chemicals with a mitogenic potential. This was tested with three presumedly nongenotoxic carcinogens in the rodent, namely cyproterone acetate (CPA), thioacetamide (TA) and phenobarbital (PB). Freshly isolated hepatocytes were cultured under 13% or 4% O2, representing the tissue oxygen tension in the periportal (13% O2) and the pericentral area (4% O2) in the liver lobules. Fluorescence intensities of QDH-stained hepatocyte nuclei of different ploidy levels (2N, 4N or 8N) were quantified by image analysis. The epidermal growth factor induced G0-G1 shift was affected by the oxygen tension and was reversible as shown after exposure to retinoic acid (RA). At subtoxic concentrations, all three nongenotoxic carcinogens induced a shift of quiescent hepatocytes of all ploidy levels into the G1 cell cycle phase within a 6-h period. CPA was the most effective compound, followed by PB and TA. The strongest induction of the G0-G1 shift was observed in most cases in 2N nuclei. The oxygen tensions applied and the individual compounds tested, differentially affected the response of the individual ploidy classes. The results correspond well with the site-specific mitogenic response within liver lobules as observed after exposure in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

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.

Albumins↗
PubMed1993

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.

Analysis of Variance↗
PubMed1993

Effects of sodium butyrate on DNA content, glutathione S-transferase activities, cell morphology and growth characteristics of rat liver nonparenchymal epithelial cells in vitro.

The effects of sodium butyrate, which has been shown to act as a differentiation promoting agent in several different tumor cell lines, were studied in a rat liver nonparenchymal epithelial cell line. Exposure of these cells to 3.75 mM butyrate resulted in an inhibition of cell proliferation and, at the same time, an increase in cell diameter (2- to 6-fold) and size of the nuclei (approximately 2-fold) after 3 days in culture. Binucleated cells arose, comprising approximately 12% of the cells investigated, and the number of cells with an abnormal set of chromosomes was increased. Intercellular communication, measured by dye transfer of Lucifer Yellow, was unchanged. From the various xenobiotic metabolizing enzyme activities measured, only those of glutathione S-transferases were significantly altered (increases of 4- to 9-fold) by butyrate treatment. These increases were mainly due to the predominant rise in the pi class isoenzyme which is a well-known tumour marker in rat hepatocarcinogenesis. Thus, our results cannot be interpreted as being either due to promotion of differentiation or due to transformation. The state and type of cell under study has to be considered and investigations of further differentiation parameters are needed to obtain a deeper insight into the biological activity and the underlying mechanisms of cell state modifying agents like butyrate.

Animals↗