Fetal Rat Liver Cells Research Articles

Monooxygenase and epoxide hydrolase regulation in primary fetal rat liver cell culture.

It is now well documented that in most instances, the production and accumulation of reactive intermediates in a living organism depend upon the relative activity of several enzymes (for review, see 1,2,3). Qualitative or quantitative modifications of these enzymatic activities might thus lead either to an enhancement or a decrease in the toxicity of a given chemical. Precise knowledge of the biochemical mechanisms which control these enzymatic systems could be of paramount importance if one wants to predict or modify the biological potential of cells to produce active metabolites.

Comparison of aryl hydrocarbon hydroxylase and epoxide hydratase. Induction in primary fetal rat liver cell culture

The activity of aryl hydrocarbon hydroxylase (AHH) and/or epoxide hydratase (EH) is induced in primary fetal rat liver cell culture by benz[ a]anthracene (BA), phenobarbital (PB), cigarette smoke condensate (CSC), 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and trans-stilbene oxide (TSO). The response of the two enzymes to the different chemicals varies as follows: (a) AHH is induced by lower concentrations of BA, PB and CSC than those required to significantly induce EH; (b) AHH is selectively induced by TCDD and by low BA concentrations; (c) the kinetics of AHH induction by BA, PB and CSC is faster than that of EH; (d) TSO is a selective inducer of EH. As described earlier for AHH, RNA and protein synthesis and the continuous presence of the inducer are required in the early phases of EH induction. Later when the EH activity has reached a plateau, intact RNA and protein synthesis is not necessary to maintain the enzyme at its optimal value. The removal of the inducer determines a decay of the EH activity, allowing the estimation of a biological t 1 2 of about 72 h. TSO prevents the AHH induction by PB, but not that mediated by BA and CSC. Added together with PB, BA, CSC or PB plus BA, TSO induces the EH activity in a more than additive manner. This effect is only seen after 6 days of continuous treatment. These results indicate that in this tissue culture model, the mechanism of AHH and EH induction can clearly be dissociated.

Establishment of a fetal rat liver cell line that retains differentiated liver functions.

Fetal rat liver cells were transformed with a temperature-sensitive A mutant (tsA255) of simian virus 40. A CLONAL CELL LINE, RLA255-4, which was temperature sensitive in the maintenance of the transformed phenotype, was isolated. This cell line expressed the transformed phenotype (rapid growth, high cell density, overgrowth of normal cells, and cloning in soft agar) at the permissive temperature (33 degrees C) and the nontransformed phenotype (slower growth, lower saturation density, decreased efficiency of overgrowth of normal cells, and lower cloning efficiency in soft agar) at the restrictive temperature (40 degrees C). The tsA255-transformed cells expressed differentiated liver functions under controllable conditions. At the permissive temperature, they produced low levels of albumin and transferrin, whereas at the restrictive temperature the transformed phenotype was lost and the production of these hepatic proteins was greatly enhanced. RLA255-4 cells contained functional receptors for glucagon, as shown by the stimulation of intracellular cyclic AMP accumulation by glucagon. The response to glucagon was dose dependent (Kact = 5 x 10(-8) M) and could be demonstrated in cells grown at both permissive and restrictive temperatures after 7 days in culture (i.e., at a cell density of approximately 4 x 10(5) cells per cm2 or higher). Addition of cortisol to the culture medium enhanced the glucagon response selectively at the restrictive temperature.

A new highly sensitive assay for ethoxycoumarin deethylase in cultured hepatocytes

Hepatocytes in culture are presently considered as an interesting model for pharmacological and toxicological investigations. The study of their cytochrome P-450-linked monooxygenases requires sophisticated methodologies to detect the usual very low enzymatic activities. In this paper, we describe a new, rapid, sensitive, and specific technique for the measurement of 7-ethoxycoumarin deethylase. This assay is based on the multiple-ion detection of the trimethylsilyl ether of 7-hydroxycoumarin. This method, developed for the study of cytochrome P-450-dependent monooxygenases in cultured fetal rat liver cells, allows the detection of activities as low as 0.2 pmol/min.

Stimulation of DNA synthesis and myo‐inositol incorporation in mammalian cells

Phosphatidylinositol (PI) synthesis and its role in controlling the cell cycle has been investigated using fibroblasts and liver cells in culture. PI synthesis as measured by incorporation of [3H]-myo-inositol into trichloroacetic acid precipitable material during 0--60 min after serum or growth factor stimulation of serum-starved cells is increased in primary fetal rat liver cells, rat embryo fibroblasts, and 3T3 mouse cells. In contrast, growth stimulation of 3T3 cells and hepatocytes rendered quiescent in G1 by amino acid starvation is not accompanied by increased incorporation of [3H[-myo-inositol into trichloroacetic acid precipitable material. This suggests that those cells might be arrested at a different point in G1 than cells arrested by serum depletion. Inhibition of PI synthesis by variation of-hexachlorocyclohexane (HCH), a steric analog of myo-inositol, during early times (e.g., 0--4 hr) after growth stimulation, reversibly blocks initiation of DNA synthesis in 3T3 cells. The results support the idea that increased PI synthesis in response to growth stimulation in the cell types studied here is a prerequisite for progression through G1 and subsequent entry into S phase.

Benzo[ a]pyrene metabolism in rat fetal hepatocytes culture. Improved methodology and effect of substrate concentration

The different characteristics of benzo[ a]pyrene (BP) metabolism in primary fetal rat liver cell culture have been investigated. We have determined the extent of the in vivo [ 3H]BP metabolism by measuring all of the metabolites retained in the cell and excreted into the culture medium. The extent of the conjugation as well as the nature of the conjugates was established and the pattern of these metabolites analyzed by high performance liquid chromatography (HPLC). The fetal hepatocytes very actively metabolize BP and readily excrete in the culture medium all the produced metabolites in the form of sulfate and glucuronide conjugates. The relative proportion of those compounds varies as a function of the substrate concentration added to the cell culture, the higher the BP concentration, the more glucuronide conjugates. The HPLC analysis of the metabolites shows that BP-1,6-quinone and -3,6-quinone are the major excreted products, indicating the probable existence of an active 6 hydroxylation reaction in the fetal hepatocytes. On the other hand, the pattern of the different metabolites is influenced by the BP concentration. At low BP doses (0.8 μM), the relative amount of polar metabolites is twice as high and that of primary phenols twice as low, when compared to those produced by cells treated with 80 μM BP. The AHH activity drastically modifies the overall rate of the BP metabolism but does not affect the qualitative pattern of the excreted metabolites. The overall metabolism of [ 3H]BP by the cell culture can easily be estimated by measuring the release of the tritiated water from the substrate into the culture medium.

The effects of leucocytic and serum factors on fibrinogen biosynthesis in cultured hepatocytes

Primary monolayer cultures of fetal rat liver cells have been used to determine whether factors derived from sera and/or leucocytes from traumatized animals can act directly on hepatocytes to cause an increased synthesis of fibrinogen, an acute-phase reactant. The data show that fibrinogen synthesis increases in a dose-response relationship by the addition of leucocytic supernatant preparations from cells of rabbits that had previously been subjected to a peritoneal injection of sterile glycogen. The response of the hepatocytes to leucocytic factor is independent of the presence of fetal bovine serum. In other experiments, fibrinogen synthesis was also significantly stimulated over controls when hepatocytes were incubated in medium supplemented with dialyzed sera from either turpentine-stimulated or sham-operated rats. These findings lend support to the notion that there is a non-dialyzable factor(s) in the sera of traumatized animals that interacts with hepatocytes to elicit the acute-phase response.

59 Regulation of aryl hydrocarbon hydroxylase and epoxide hydratase activity in primary fetal rat liver cell culture

59 Regulation of aryl hydrocarbon hydroxylase and epoxide hydratase activity in primary fetal rat liver cell culture

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 E 1 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.

Lack of a correlation between polyamine synthesis and DNA synthesis by cultured rat liver cells and fibroblasts.

Growth stimulation of either fetal rat liver cells or rat embryo fibroblasts in culture results in considerable increases in intracellular polyamine levels as cells proceed through the cell cycle. Treatment of such cell cultures with appropriate levels of two inhibitors of polyamine synthesis, namely alpha-hydrazino ornithine and methylglyoxal bis (guanylhydrazone), can essentially completely block these increases in cellular polyamine content. Under such conditions, where the elevation in intracellular polyamine content is prevented, cell cultures are nevertheless able to initiate DNA synthesis and subsequently synthesize DNA at rates comparable to untreated control cultures that have been growth-stimulated. These two cell types therefore contain sufficient polyamines when in a resting state (G1) to enable them to enter from G1 into S phase and traverse S phase at normal rates in the absense of further polyamine synthesis. The recruitment of cells into the first cell cycle, through serum stimulation of growth, therefore appears not to be mediated or regulated by the increases in intracellular levels of polyamines that occurs under these conditions. Conversely, the arrest of growth of these cell types resulting from serum deprivation is not mediated by a limitation of intracellular polyamine content.

Sedimentation velocity distributions of cells from adult and fetal rat liver and ascites hepatoma

Cells isolated from adult and fetal rat liver and ascites hepatoma were separated into distinct populations by velocity sedimentation at unit gravity. Normal adult liver ceils sediment with modal velocities ranging from 5 to 50 mm/h. Volume analysis using a Coulter-type counter demonstrated that the separation was based primarily on cell size. Appreciable differences were observed in the sedimentation velocity distribution of cells isolated from different normal lobes or regenerating liver. Most fetal rat liver cells sediment with velocities inferior to 12 mm/h. Ascites (Novikoff) hepatoma cells present a velocity distribution more similar to that of fetal than to normal adult liver cells. The results are discussed in terms of cell-size changes associated with liver maturation, regeneration or transformation.

Growth of fetal rat liver cells in response to hydrocortisone

Growth of fetal rat liver cells in response to hydrocortisone

Growth control in primary fetal rat liver cells in culture

Primary fetal rat liver cells cultured in medium deficient in, but not free of, arginine in the presence of dialyzed fetal calf serum grow until the final cell density is attained and cells become quiescent in the Go phase of the cell cycle. When growing cells are transferred into arginine free medium, cells become reversibly arrested in Go. Fetal rat liver cells can be induced to synthesize DNA by addition of high levels of arginine to serum free medium. Low arginine levels in the culture medium do not induce cell growth unless serum is present. Serum stimulates arginine uptake in fetal rat liver cells suggesting that serum growth factor(s) act by increasing intracellular arginine levels high enough to initiate the growth cycle. Fractionation of fetal calf serum by gel filtration on G-200 Sephadex yields a partially purified arginine uptake stimulating activity which is eluted from the column in the same fractions that contain fetal rat liver cell growth promoting activity. Insulin induces DNA synthesis in quiescent fetal rat liver cells. Glucagon reverses the stimulatory effects of insulin. N-6,O-2-Dibutyryl adenosine 3:5-cyclic monophosphoric acid (But2c-AMP) (10-minus4 M) and theophilline (10-minus3 M) inhibit arginine uptake and the initiation of DNA synthesis by serum. The role of arginine in the control of DNA synthesis in fetal rat liver cells and the mechanism of action of serum growth factors are discussed.

Toxicity of dieldrin to primary culture of rat fetal liver cells and human kidney cell line

Rat fetal liver primary culture cells (RLP) were exposed to 25–200 μg dieldrin/ml medium for 48 hr. The morphologic changes observed included granulation and shrinkage of the cytoplasm, formation of long and narrow cytoplasmic projections, and appearance of giant cells. At the higher concentrations the morphologic changes were noted within a few hours after exposure to the pesticide, and a considerable number of cells were separated from the monolayer. At the 25μg/ml level there was some decrease in the number of viable cells and a reduction in the total protein, DNA, and RNA contents of the cells. All the chemical and morphologic effects of the challenge were dose related. Concentrations above 100 μg/ml brought about a complete destruction of the cell monolayer. Human kidney cell line B culture was slightly less sensitive than the RLP cells, but demonstrated similar morphologic alterations when challenged by slightly higher dieldrin concentrations.

Studies on the control of pyruvate oxidation in isolated fetal rat liver cells

The control of pyruvate oxidation in isolated fetal rat liver cells has been studied by incubating these cells with glucose, in the absence and in the presence of uncouplers of oxidative phosphorylation. 1. 1. It was found that cells suspended in Krebs-Ringer-phosphate medium accumulated lactate and pyruvate under aerobic conditions. The lactate/pyruvate ratio was found to be 7.5 in the presence of glucose, increasing to 70, 193 and 48 in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), valinomycin and nigericin, respectively. Measurements of glycolytic intermediates and adenine nucleotides showed that the changes in the lactate/pyruvate ratio correlated with changes in the phosphate potential and the 3-phosphoglyceric acid/glyceraldehyde 3-phosphate ratio, according to the equation: [ pyruvate] [ lactate] = K ldh K gapdh · K pgk · [ ATP] [ ADP] · [ P i ] · [3- phosphoglyceric acid] [ glyceraldehyde 3- phosphate] where K ldh , K gapdh and K pgk are the equilibrium constants of lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. 2. 2. The flux through pyruvate dehydrogenase has been measured with [3,4- 14C 2]glucose. In the absence of uncouplers, this flux was found to be 0.4 nmole/min per mg protein, increasing to 0.6 and 0.9 nmole/min per mg protein in the presence of FCCP and valinomycin, respectively. Measurement of the specific activity of lactate permitted the calculation that 90% of the pyruvate formed from glucose in the absence of uncouplers is reduced to lactate and only 10% is oxidized by the mitochondria. Intraperitoneal injection of pregnant rats with thiamine increased the percentage of pyruvate oxidized by the mitochondria of the fetal liver cells to 30. It is concluded that in fetal liver cells pyruvate oxidation is controlled at the level of pyruvate dehydrogenase.

Lack of effect of erythropoietin on cyclic adenosine-3',5'-monophosphate levels in rat fetal liver cells.

Abstract The effect of Erythropoietin (Ep) and epinephrine on the cellular concentration of adenosine 3′,5′-cyclic monophosphate (cAMP) was studied using suspensions of fetal liver cells obtained from 14-day pregnant rats. This system was chosen because the cell suspensions were greater than 65 per cent erythroid and very sensitive to Ep. In cells preincubated with theophylline, Ep failed to increase cAMP levels after both 5 minute and 30 minute incubations with the hormone. By contrast, epinephrine stimulated cAMP concentrations 6- to 7-fold indicating that the adenyl cyclase present was capable of responding to an appropriate stimulus. In suspensions first treated with epinephrine to raise cAMP levels, addition of Ep did not lower the concentration of this nucleotide. Heme synthesis experiments employing the same conditions as the cAMP studies show that Ep effectively stimulated 59 Fe incorporation into heme. This indicated that under the conditions of the cAMP studies the hormone was fully capable of interacting with the erythropoietin-responsive cell. The above experiments show that erythropoietin can stimulate hemoglobin synthesis in vitro without producing any detectable increase or decrease in the cellular cAMP concentration. Thus, the data fail to support the hypothesis that the effect of Ep on red-cell production is mediated by a change in cAMP levels.

Aryl Hydrocarbon Hydroxylase Induction in Mammalian Liver Cell Culture: III. EFFECTS OF VARIOUS SERA, HORMONES, BIOGENIC AMINES, AND OTHER ENDOGENOUS COMPOUNDS ON THE ENZYME ACTIVITY

Abstract Aryl hydrocarbon hydroxylase induction in fetal rat liver cells in culture by phenobarbital or 3-methylcholanthrene proceeds just as well in 10% heat-inactivated, boiled, or dialyzed calf serum as in regular 10% calf serum in the growth medium. After the hepatocytes are attached, the serum-containing medium can be replaced with serum-free, completely defined medium for several days, so that the influence of various hormones or other endogenous compounds on the basal and inducible hydroxylase activities can be evaluated. Significant stimulatory effects are produced by testosterone, glucagon, and prostaglandins; significant decreases are seen with 17-β-estradiol, insulin, and dexamethasone. No significant effects are found with adrenocorticotropic hormone, human growth hormone, thyroxine, prolactin, derivatives of cyclic adenosine 3',5'-monophosphate, and numerous other endogenous compounds such as fatty acids and alkyl polyamines, alone and in combinations. The kinetics of aryl hydrocarbon hydroxylase induction by 1.0 mm norepinephrine or by other biogenic amines of similar structure is the same as that by phenobarbital or 3-methylcholanthrene. The effects of phenobarbital, 3-methylcholanthrene, and biogenic amines with either a phenyl or indolen ucleus on the hydroxylase induction are additive or synergistic when two or three of these types of inducers are combined in the culture medium. From the use of an aromatic l-amino acid decarboxylase inhibitor and a monoamine oxidase inhibitor added prior to the biogenic amines, we conclude that the amine, rather than any metabolite, is directly involved in causing hydroxylase induction. The effect of norepinephrine is greater than that of phenobarbital or 3-methylcholanthrene in retarding the normal rate of degradation of induced hydroxylase activity in the presence or the absence of protein synthesis. Either phenobarbital or 3-methylcholanthrene or norepinephrine as a second inducer can direct at some posttranscriptional level a further rise in hydroxylase activity after treatment of the hepatocytes with the first inducer.

Studies on primary cultures of differentiated fetal liver cells.

A method for culturing non- or slowly growing, differentiated fetal rat liver cells is described. It involves the use of collagenase as a digesting agent and of a selective medium deficient in arginine which suppresses the growth of nonparenchymal liver cells. Evidence is presented that surviving cells (a) retain liver-specific urea cycle functions measured by their capacity to transform ornithine into arginine, (b) synthesize DNA in glucose-deficient medium, and (c) synthesize and secrete albumin. This primary cell culture responds to partially hepatectomized rat serum and may be an appropriate assay system for the study of mechanisms which regulate liver regeneration.

Stimulation of DNA and protein synthesis in fetal-rat liver cells by serum from partially hepatectomized rats.

Primary cultures of differentiated liver cells from fetal rats respond to serum from partially hepatectomized rats by an increased uptake of [(3)H]thymidine and [(3)H]leucine as compared with cultures incubated with normal rat serum. These findings are discussed in relation to the mechanism of induction of DNA synthesis and growth in liver cells during liver regeneration.

A morphometric and biochemical study of fetal and adult rat liver cells, with special reference to energy metabolism.

A morphometric and biochemical study of fetal and adult rat liver cells, with special reference to energy metabolism.