Genetic analysis of aflatoxin B1 activation in rat hepatoma cells

Summary We present a strategy to elucidate the rate-limiting steps in activation of carcinogenic compounds by cytochromes P450. The principle was to select Reuber rat hepatoma cells for resistance to a procarcinogen. The hypothesis was that resistant cells should be systematically deficient in the P450 enzyme(s) involved in the activation process. Here we present an example of the use of this approach using aflatoxin B1 (AFB1), a potent hepatocarcinogen, as the selective agent. Parental cells as well as individual and pooled colonies selected for AFB1 resistance from three independent rat hepatoma lines were characterized for their content of 1) mRNA hybridizing to cDNA and/or oligonucleotide probes for cytochromes P450IIB1, P450IIB2 and albumin; and 2) aldrin epoxidase activity. Parental aflatoxin B1-sensitive cells were shown to express P450IIB1 but not P450IIB2. The majority of the aflatoxin B1-resistant clones failed to accumulate cytochrome P450IIB1 mRNA and expressed no or only very low aldrin epoxidase activity. Albumin mRNA levels remained unchanged, demonstrating that loss of expression of cytochrome P450IIB1 was not a consequence of a general dedifferentiation event. A revertant population showing restoration of both cytochrome P450IIB1 mRNA accumulation and aldrin epoxidase activity was fully sensitive to aflatoxin B1. The correlation between expression of cytochrome P450IIB1 and sensitivity to aflatoxin B1 in both parental cells and revertants strongly suggests that cytochrome P450IIB1 is a major contributor to the activation of aflatoxin B1 in rat hepatoma cells. The kind of strategy described here could be applied to other compounds that become cytotoxic for hepatoma cells following activation by cytochromes P450.Abbreviations AFB1 aflatoxin B1 - AE aldrin epoxidase - AHH aryl hydrocarbon hydroxylase - PAH polycyclic aromatic hydrocarbons - PB phenobarbital     

Enhancement by streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine of the tyrosine aminotransferase activity in cultured rat liver cells: role of dexamethasone and NAD

The activity of tyrosine aminotransferase (TAT) (EC 2.6.1.5) was enhanced 3-fold after a 5-h exposure of cultured rat liver cells (RLC) to streptozotocin (SZ) at concentrations higher than 100 microgram/ml (0.38 mM) in the presence of 10 nM dexamethasone, a potent glucocorticoid inducer for the enzyme. The structurally related carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also enhanced the aminotransferase in the presence of the glucocorticoid, but its optimal concentration was at 100 ng/ml (0.68 microM). While the cellular NAD (NAD+ + NADH) concentration was reduced to 60% of the control levels, the rate of poly(ADP-ribose) formation in the isolated cell nuclei was unaffected by treating the cells with SZ. The enhancement of tyrosine aminotransferase by SZ and MNNG was effectively prevented by nicotinamide. Using nicotinamide and its derivatives such as 1-methyl-, N'-methyl- or 6-amino-derivatives it was found that the degree of enzyme induction is almost inversely proportional to the cellular NAD content, though the activity of nuclear poly(ADP-ribose)polymerase remains unchanged. The results indicate that SZ or MNNG, in combination with dexamethasone, stimulate the induction of tyrosine aminotransferase through their NAD lowering action.     

Activation of protein kinase C sensitizes the cyclic AMP signalling system of T51B rat liver cells

Activation of protein kinase C (PKC) bu phorbol esters (TPA) results in a modification of the cyclic AMP system leading to either attenuation or amplification of the cyclic AMP signal. In the non-neoplastic T51B rat live cell line, TPA, when added to intact cells, had no effect on the basal level of cyclic AMP synthesis but caused a 1.5 fold amplification of the stimulation induced by β-adrenergic agents, cholera toxin and forskolin. The effect appeared to be mediated by PKC since diacylglycerols caused the same amplification as did TPA while inactive phorbol esters were without effect. Phosphorylation of Gs or the catalytic subunit of adenylate cyclase by PKC is likely to be responsible for the enhancement of cyclic AMP synthesis. TPA also caused translocation of PKC; however, the time course of the translocation was loner than the time course of the enhancement of adenylate cyclase activity. Thus, the ability of TPA to amplify cyclic AMP synthesis is probably mediated by activation of PKC that is already present in the membrane.     

Characterization of rat liver cells transformed in culture bydl-ethionine

Summary A rat liver-derived epithelial cell line transformed withdl-ethionine and the corresponding control cell line were characterized according to morphological and cytochemical criteria to establish their origin from liver epithelium and to identify cellular changes due to transformation bydl-ethionine. The presence of intermediate junctions confirms the epithelial nature; glycogen accumulation and glucose-6-phosphatase activity confirm the hepatic origin of the cells. Persistent alterations resulting from ethionine transformation were variations in cell shape and size, focal multilayered growth, an increase in the nucleolar: nuclear ratio, and a reduction in the number of cells displaying a primary cilium. Hyperplasia of the inner nuclear membrane, elongation and branching of mitochondria, and a reduction in the length and frequency of cell junctions were also characteristic of the transformed cells.     

Intracellular aflatoxin B1-binding proteins in rat liver

Intracellular aflatoxin B1 binding in rat liver was studied under both in vitro and in vivo conditions. Binding in vivo appeared similar to that observed in vitro except that some covalent adduct formation was detected. Participation of previously described carcinogen-binding proteins such as the Ah receptor, h2-5S protein, 4-5S receptor for 3-methylcholanthrene and the Z-protein fraction was discounted on the grounds of competition binding studies and gel-permeation chromatography. The molecular weight of 45,000 was estimated for the major aflatoxin B1-binding component. Aflatoxin B1 co-eluted with the glutathione S-transferases during gel-permeation and separation of the various isozymes by cation-exchange chromatography indicated interactions with the YaYa and YaYc-forms. These proteins, however, account for less than 20% of the total intracellular aflatoxin binding. A protein of apparent monomeric structure appears to form the major in vitro/in vivo complex with aflatoxin B1.     

Factors regulating the induction of ornithine decarboxylase in fetal rat liver cells in culture

Various hormonal and non-hormonal agents were tested for their ability to induce ornithine decarboxylase (EC 4.1.1.17) in primary cultures of fetal rat liver cells that retain many of the differentiated functions of hepatocytes. The only agents to induce ornithine decarboxylase in this cell type were fetal calf serum, prostaglandin E₁ and cyclic AMP derivatives. Also, the amino acid arginine would induce ornithine decarboxylase in this cell type following arginine starvation for 24 h. These observations are in contrast to the wide range of hormones, e.g. insulin, hydrocotisone, glucagon and growth hormone, that can induce ornithine decarboxylase in vivo in the adult rat liver but which are all without effect on fetal rat liver cells.     

基于IPA分析自噬对大鼠肝再生中树突状细胞的调节作用

为探讨自噬对大鼠肝再生中树突状细胞(Dendritic cells, DCs)的调节作用,文章通过Percoll 密度梯度离心结合免疫磁珠分选分离大鼠DCs,Rat Genome 230 2.0芯片检测大鼠肝再生中自噬相关基因表达变化,利用IPA等软件分析自噬在DCs中的生理活动。结果表明,LC3、BECN1、ATG7和SQSTM1等关键基因在部分肝切除后不同恢复时间段有明显表达变化;芯片中对应的自噬相关基因为593个,其中210个基因发生了有意义的变化。比较分析自噬生理活动情况,发现自噬在再生早期和晚期阶段增强,增殖期减弱。与自噬相关的生理活动主要有RNA表达、RNA转录细胞分化和增殖,其中涉及的信号通路主要有PPARα/RXRα激活、急性期反应、TREM1 信号通路、IL-6 信号通路、IL-8 信号通路和IL-1 信号通路等,它们在肝再生阶段发生了不同程度的上调或下调。Cluster 分析还发现,P53和AMPK信号参与调控DCs的自噬活动,在肝再生早期主要是AMPK信号,在肝再生末期P53和AMPK信号共同参与自噬的调节。以上研究结果说明DCs自噬可能在肝再生早期激活细胞免疫反应和后期清除DCs等方面发挥着重要作用。     

Modulation of cell proliferation and polyamine metabolism in rat liver cell cultures by the iron chelator O-trensox

The antiproliferative effects of the iron chelator O-trensox and the ornithine-decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) were characterized in the rat hepatoma cell line FAO, the rat liver epithelial cell line (RLEC) and the primary rat hepatocyte cultures stimulated by EGF. We observed that O-trensox and DFMO decreased cell viabilty and DNA replication in the three culture models. The cytostatic effect of O-trensox was correlated to a cytotoxicity, higher than for DFMO, and to a cell cycle arrest in G0/G1 or S phases. Moreover, O-trensox and DFMO decreased the intracellular concentration of spermidine in the three models without changing significantly the spermine level. We concluded that iron, but also polyamine depletion, decrease cell growth. However, the drop in cell proliferation obtained with O-trensox was stronger compared to DFMO effect. Altogether, our data provide insights that, in the three rat liver cell culture models, the cytostatic effect of the iron chelator O-trensox may be the addition of two mechanisms: iron and polyamine depletion.     

Content of dolichol and retinol in isolated rat non-parenchymal liver cells

The liver sinusoids, that are considered as a functional unit, harbour four types of sinusoidal cells (Ito, Kupffer, endothelial and pit cells). Dolichol content has been determined in many tissues and subcellular compartments, alteration has been reported in many types of liver injury, but until now no data are available on its content in every type of sinusoidal non-parenchymal liver cells. Dolichol and retinol metabolism might intersect in their traffic in biological membranes. Intercellular as well as intracellular exchange of retinoids is an essential element of important processes occurring in liver cells. It has been suggested that the role of dolichol, besides being a carrier of oligosaccharides in the biosynthesis of N-linked glycoproteins, may be to modify membrane fluidity and permeability, and facilitate fusion of membranes. Dolichol in the membrane is intercalated between the two halves of the phospholipid bilayer, but its exact disposition is not known and the movement and distribution of retinoid in membranes may vary with the geometry of the membranes. Therefore the aim of this study is to obtain a global understanding of the sinusoidal system regarding dolichol and retinol content in each type of isolated rat liver sinusoidal cell, in normal conditions and after vitamin A administration. The information that can be drawn from the present results is that with normal vitamin A status of the animal, the dolichol content is almost uniform in all liver cells. After vitamin A supplementation, a great increase of dolichol, together with the known increase of retinol, can be measured only in a subpopulation of the Ito cells, the Ito-1 subfraction. Therefore in the cells that are present in the hepatic sinusoid, different pools of dolichol may have separate functions. Because retinol traffic among cells, membranes and plasma still remains to be fully understood, roles of dolichol in the exchange of vitamin A among sinusoidal liver cells are discussed. © 1998 John Wiley & Sons, Ltd.     

The in vivo disposition of aflatoxin B1 in rat liver

The disposition of a non-toxic i.p. dose of [3H]-aflatoxin B1 (0.70 micrograms/kg) in the blood, plasma, and liver was studied in male Wistar rats. Uptake into the blood, plasma, and liver was biphasic; there was an initial rapid rise (0-2 hr) followed by a second phase (2-12 hr) of a gradual increase. Most of the radioactivity in the blood was bound noncovalently to albumin. Distribution of radioactivity in the subcellular fractions of liver showed that the microsomes exhibited the highest labeling which increased over the time course; labeling of the cytosol reached a maximum at 2 hr then decreased to a new steady state, whereas the mitochondria and nuclei reached a plateau. When the content of aflatoxin B1 in the nuclear subfractions was examined, greater than 92% of the total radioactivity was found in the deoxyribonucleoprotein fraction, and 84% of this was bound noncovalently. These results suggest that aflatoxin B1 is transported from the site of injection through the blood to the liver and its subcellular and subnuclear fractions primarily in a noncovalent form.     

Primary monolayer cultures of adult rat liver parenchymal cells suitable for study of the regulation of enzyme synthesis

Summary Parenchymal cells from adult rat liver which had been fully regenerated were isolated and cultured in nonproliferating monolayers in vitro. The optimum conditions for attachment of these cells to Falcon plastic dishes were determined. When approximately 1.0×10⁵ nuclei per cm² suspended in Ham's F-12 medium with 0.5 μg of insulin per ml and 25% fetal calf serum were incubated at 37°C for 24 hr, about 50% became attached and contiguous. When the above medium was supplemented with synthetic buffers 2-(N-morpholino) ethanesulfonic acid (MES) andN-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), the presence of 15% fetal calf serum also allowed an attachement effiency of 50%. Tyrosine aminotrasferase activity in the cells was elevated when the culture medium was supplemented with hydrocortisone or dexamethasone. The largest increases were observed after 72 hr of culture. Cycloheximide prevented the increase. Presented in part at the 24th Annual Meeting of the Tissue Culture Association, Boston, Mass. June 4 to 7, 1973. The work was supported in part by National Cancer Institute Grants CA-51304-01 (R. J. B.) and CA-07175. P. R. W. was a Damon Runyon Memorial Fund Postdoctoral Fellow.     

Hepatoblast and mesenchymal cell-specific gene-expression in fetal rat liver and in cultured fetal rat liver cells

The aim of this study was to determine whether passaged rat fetal liver cells are functional hepatoblasts. Hepatocyte/hepatoblast- and liver myofibroblast-gene-expressions were studied in adult and fetal rat liver tissues as well as in primary and passaged cultures of isolated rat fetal liver cells at both the mRNA and protein level. Desmin- and Alpha-Smooth Muscle Actin (SMA)-positive cells were located in the walls of liver vessels, whereas Desmin-positive/SMA-negative cells were distributed within the liver parenchyma. Primary cultures contained Prox1-positive hepatoblasts, Desmin/SMA-positive myofibroblasts and only a few Desmin-positive/SMA-negative cells. Albumin and alpha-fetoprotein (AFP) could be detected in the primary cultures and to a lesser extent after the first passage. The number of Desmin-positive/SMA-negative cells decreased with successive passage, such that after the second passage, only Desmin/SMA-positive cells could be detected. SMA-gene-expression increased during the passages, suggesting that myofibroblasts become the major cell population of fetal liver cell cultures over time. This observation needs to be taken into account, should passaged fetal liver cells be used for liver cell transplantation. Moreover it contradicts the concept of epithelial-mesenchymal transformation and suggests rather that selective overgrowth of mesenchymal cells occurs in culture. Tümen Mansuroglu and József Dudás contributed equally to this work.     

Functional changes in rat liver tRNA following aflatoxin B1 administration

Administration of aflatoxin B₁ (3 mg/kg body wt) to rats leads to strong inhibition of the acceptor activity of liver tRNA as measured by charging with [¹⁴C]-chorella protein hydrolysate. The maximum inhibition occurs 2 h after treatment. At increasing intervals after treatment, the inhibition appears to be gradually relieved, till control values are restored by 72 h. The charging experiment using several [¹⁴C]-amino acids separately shows pronounced inhibition of acceptor activity of all tRNA species, although the degree of inhibition varies with individual species. Preliminary results seem to rule out the possibility of hypermethylation of tRNA or damage to the CCA terminus as probable causes. The resultant functional changes may be attributed to a covalent interaction of aflatoxin B₁-metabolite with tRNA.     

Activation of aflatoxin B1 by a mono-oxygenase system localized in rat liver mitochondria

Structurally intact rat liver mitoplasts free of detectable microsomal contamination contain enzymatic activity to metabolize aflatoxin B1 (AFB1). The activated component(s) bind to mitochondrial macromolecules and also inhibit mitochondrial protein synthesis. The activity of intact mitoplasts or sonicated particles is partly dependent on the addition of NADPH-generating system. Under optimal conditions, the mitochondrial enzyme has specific activity of 60 to 65 pmol/mg and represents about 15 to 18% of total cytoplasmic activity for AFB1 activation. The enzyme is localized in the soluble fraction of mitochondrial matrix and appears to be distinctly different from the microsomal activity.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.