Cultures Of Fetal Hepatocytes Research Articles

The development of phenylalanine hydroxylase in rat liver; in vivo, and in vitro studies utilizing fetal hepatocyte cultures

Abstract Phenylalanine hydroxylase (PAH) is first detected in the liver of 21-day-gestation rats. Activity increases after birth, and in 10-day-postnatal rats it is about equal to that observed in the adult. The developmental pattern for the enzyme is reflected in the level of its mRNA determined by hybridization to 32P-cDNA, which is specific for PAH. Studies with cultured adult hepatocytes reveal that the addition of dexamethasone and dibutyryl cAMP to the medium maximizes the yield of enzyme. Hepatocytes derived from 21-day-gestation rats will produce enzyme in cultures maintained in medium supplemented with dexamethasone and dibutyryl cAMP. However, less mature cells, taken from 19-day-gestation rats do not produce measurable levels of enzyme activity. The relative amounts of PAH mRNA in the respective cultures reflect the level of PAH activity. Interestingly, after 3 days of culture, 19-day-gestation hepatocytes can be shown to express PAH mRNA. Therefore, with respect to the expression of PAH, we conclude that 19-day-gestation liver cells will differentiate during culture.

Long-term culture of fetal rat hepatocytes in media supplemented with fetal calf-serum Ultroser SF or Ultroser G.

Fetal hepatocytes cultured in medium supplemented with fetal calf serum (FCS) or Ultroser SF do not maintain production of albumin or transferrin beyond one week of culture. When dexamethasone (10(-7) M) is present, secretion of albumin and transferrin can be extended to two weeks, however, levels are extremely low. By three weeks, neither plasma protein can be detected in the culture medium in either conditions of culture. In contrast, hepatocytes maintained in medium supplemented with Ultroser G continue to produce albumin and transferrin at high levels for the entire three week period of this study. The morphology of the cultures are different. In FCS and Ultroser SF supplemented medium there are many more fibroblast and epithelial-like cells and relatively fewer cells which are distinctly hepatocytes when compared with Ultroser G supplemented medium. The level of tyrosine aminotransferase, which is a dexamethasone inducible enzyme, is found to be much higher in Ultroser G cultures, with no further increase demonstrable by addition of dexamethasone. In contrast, dexamethasone induces the enzyme by about eight-fold in cultures maintained in FCS supplemented medium. Therefore it appears that Ultroser G already contains sufficient steroid activity to maximize the level of tyrosine aminotransferase. A comparison between Ultroser C and SF (steroid-free) suggests that the mixture of steroid and steroid derivatives in the G formulation must be important in the maintenance of differentiated functions of hepatocytes in culture. However, supplementation of FCS cultures with dexamethasone, which is known to be present in Ultroser G, does not allow hepatocytes to retain their differentiated functions over an extended period. Therefore it is concluded that other components besides dexamethasone must be important.

Rat corticosteroid-binding globulin (CBG) biosynthesis by fetal hepatocytes in culture

The ability of fetal liver to synthesize and secrete corticosteroid-binding globulin (CBG) was investigated using primary cultures of hepatocytes transplanted from 15- and 18-day-old rat fetuses. Analysis of culture medium, after labelling with [ 14C]leucine, by crossed immunoelectrophoresis, clearly demonstrated CBG, albumin and alpha-fetoprotein (AFP) secretion. The relative rate of CBG secretion was more important at the younger stage. Indirect immunofluorescence permitted localization of CBG in fetal hepatocytes. The results suggest that the high level of CBG found in fetal serum is mainly produced by the liver of the fetus itself.

Induction by 9-hydroxyellipticine of aryl hydrocarbon hydroxylase in perinatal rat liver and in primary fetal hepatocytes in culture

In neonatal and, to a lesser extent, in fetal rat liver, 9-hydroxyellipticine was able to promote the induction of cytochrome P-450, supporting especially aryl hydrocarbon hydroxylase (AHH) but not aldrin epoxidase activity. The examination of benzopyrene metabolites by high performance liquid chromatography (HPLC) or by benzopyrene-DNA adducts formation shown that, as in adult animals, the formation of hydroxylated metabolites in position 9,10 was enhanced. In primary fetal liver cells culture, similar effects were observed. Furthermore, the presence of glucocorticoids in the culture medium was not required for the induction of AHH by 9-hydroxyellipticine (9-OHE).

Human fetal liver cultures: Basal activities and inducibility of epoxide hydrolases and aryl hydrocarbon hydroxylase

The environmental influence of various drugs on the epoxide hydrolase with styrene oxide (EH so) or benzo(a)pyrene-4,5-oxide (EH BPox) as substrate and the aryl hydrocarbon hydroxylase (AHH) activity was studied in monolayer cultures of human fetal hepatocytes (HFH) obtained at legal abortions. Hepatocytes were isolated by trypsin treatment of liver fragments and primary HFH cultures were maintained in Eagle's minimum essential medium supplemented with 15% newborn calf serum. The HFH were plated on culture dishes and allowed to ‘settle’ for one day before adding various drugs (in 1μl dimethylsulfoxide/ml) or solvent only and assay 1–2 days later. The basal AHH activity [assayed with 3H-benzo(a)pyrene as substrate] varied between 2 and 8.4 pmoles/min/mg protein and the basal EH so activity was 0.3–4.9 nmoles/min/mg protein ( n = 6) after one or two days' culture. The corresponding activity of EH BPox was 0.23–1.48 nmoles/min/mg protein ( n = 5). Exposure of cultures to 2 mM phenobarbital (Pb), 2.5–25.0 μM benzanthracene (BA), 0.1 mM trans-stilbene oxide (TSO), or 5 μM β-naphtoflavone (βNF) resulted in a 1.2–3.7-fold induction of EH so. Induction of EH BPox was also observed with Pb, βNF, BA and TSO as inducers. Pb gave a dose-dependent induction of both EH at 0.1, 1.0 and 2.0 mM. Our results demonstrate that EH and AHH activities in HFH cultures are inducible by classical in vivo inducers. Although difficult to prove, it is plausible that such induction takes place also in intrauterine life.

The expression of different monooxygenases supported by cytochrome P-450 in neonatal rats and in primary fetal hepatocytes in culture.

In rat liver, the perinatal development of various monooxygenase activities follows different patterns, depending upon the reaction studied. The ontogeny of the 6 beta-, 7 alpha- and 16 alpha-testosterone hydroxylase activities differs very significantly. Aldrin epoxidase and steroid-metabolizing monooxygenases are expressed in primary fetal rat liver cells in culture after treatment in vitro with dexamethasone. Testosterone is not metabolized by the control cells and is hydroxylated on the 6 beta and 16 alpha positions following the addition of corticoids to the culture medium. The dose and time curves vary according to the hydroxylated position of the steroid. Aldrin epoxidase activity is nearly undetectable in the control cells, but is present and is inducible by phenobarbital following treatment with the corticoid. Phenobarbital induces aldrin epoxidase in the absence of dexamethasone in the culture medium, providing that the cells are pretreated with the corticoid for 48 h. The use of antibodies against the main cytochrome P-450 species purified from adult and phenobarbital-treated rats confirms that a similar cytochrome P-450 can be induced in fetal cells in culture. The perinatal regulation of biological events, such as the expression of the monooxygenases, can be reproduced in fetal rat liver cells in culture; such a model constitutes a unique tool for studying the biochemical mechanisms which control these phenomena.

Induction of cytochrome P‐450‐dependent drug metabolism by juvenile hormone mimics in mammalian cell culture

AbstractThe monooxygenase activity of fetal hepatocytes in culture shows a differential response toward juvenile hormone I and analogs. Juvenile hormone I, R‐20458, and Methoprene increase the deethyiation of 7‐ethoxyresorufin while not affecting or even inhibiting the N‐demethylation of p‐chloro‐N‐methylaniline. RO‐203600, a 1,3‐benzodioxole‐containing analog, increases both the deethylase and the N‐demethylase, whereas Hydroprene does not affect either activity. The inductive effect with juvenile hormone I is obtained with exposure periods of at least 30 min and is maximum when the concentration of the hormone is 14 μM in the medium. This amount results in the covalent binding to cellular macromolecules of 1.3 × 19−18 moles/cell. The induction requires continuous protein synthesis but RNA synthesis only for a short initial period. It is concluded that juvenile hormone and mimics induce specific cytochrome P‐450 species in fetal liver cells even if the culture conditions are not optimal. The toxicological implications of these results are briefly discussed.

Effects of basement membrane matrix on the culture of fetal mouse hepatocytes.

The effects of laminin, fibronectin and type IV collagen, all of which are major matrix components of the basement membrane, upon fetal mouse hepatocyte culture were studied. Among these matrices, laminin showed the greatest effect on DNA synthesis, cell proliferation, the secretion of alpha-fetoprotein and cell attachment. The effects of fibronectin and type IV collagen were slight with regard to the promotion of growth and cell attachment. However, there were no differences in the secretion of albumin into the media among the groups. These results suggest that laminin may be necessary for the cellular growth and functional maintenance of immature liver cells during normal development of the liver in vivo.

Primary cultures of fetal hepatocytes from the genetically obese Zucker rat: protein synthesis.

Primary fetal hepatocytes derived from Zucker rats with expected fa gene frequencies of 0.0 and 0.75 have been established and can be used to detect early effects of the fa gene on hepatocellular metabolism. Paired incubation experiments demonstrate that protein synthesis in 0.75 fa gene cultures is significantly less than in 0.0 fa gene cultures under basal conditions. Insulin stimulates protein synthesis in 0.0 fa gene cultures but has no effect on 0.75 fa gene cultures. Cycloheximide inhibits protein synthesis in both types of culture. NH4Cl inhibits protein synthesis in 0.0 but not in 0.75 fa gene cultures. These data suggest that fetal hepatocytes bearing the fa gene have in vitro a generally sluggish anabolic capacity and a blunted capacity to respond to insulin compared to fetal hepatocytes without the fa gene. These diminished capacities may be expression of a genetic error in lysosomal function.

Multiplicity of cytochrome P-450 in primary fetal hepatocytes in culture.

Primary fetal rat hepatocytes in culture display different monooxygenase activities which can be induced by several chemical inducers. Until now, these hepatocytes were believed to produce only one single cytochrome P-450 species, namely the cytochrome P1-450 (or P-448). It now seems possible to induce other cytochrome P-450 species in these hepatocytes, providing that they receive an appropriate hormonal treatment. We have examined the effect of dexamethasone on the cytochrome P-450 type supporting different enzymic activities (aryl hydrocarbon hydroxylase, ethoxycoumarin deethylase, aldrin monooxygenase). Our results show that the presence of dexamethasone in the culture medium produces qualitative and quantitative changes in the monooxygenase-supporting cytochrome(s) P-450. For low dexamethasone concentrations (between 1 nM and 0.1 microM) a cytochrome P-450 is formed displaying biochemical and biophysical properties similar to those induced by phenobarbital in the adult rat liver. At higher concentrations (above 1 microM), similar qualitative changes are observed; but a quantitative phenomenon occurs, the (cytochrome P-450)-dependent enzymic activities being also induced. Dexamethasone also has a synergistic effect on the induction of enzymic activity by the mixture of phenobarbital plus benzanthracene. The various biochemical changes induced by dexamethasone in the fetal cell cultures parallel those observed in vivo during the perinatal period of life. The cell culture system may thus constitute an interesting model for studying the ontogenic development of liver monooxygenases.

Primary cultures of fetal hepatocytes from the genetically obese Zucker rat: characterization and total lipogenesis.

Primary fetal hepatocyte cultures derived from Zucker rats and with expected fa-gene frequencies of 0.0 and 0.75 have been established and can be used to detect early effects of the fa gene on hepatocellular metabolism. Proliferative capacity is similar in both types of culture. Changes of the growth media significantly decrease total lipogenesis in both 0.0 and 0.75 fa-gene culture grown in arginine-free DME medium. Paired incubation experiments demonstrate that total lipogenesis in 0.75 fa gene cultures is significantly less than in 0.0 fa-gene cultures under basal conditions. Stimulation of total lipogenesis by pharmacological doses of insulin and excess substrate (glucose) is significantly less in the 0.75 fa gene than in the 0.0 fa-gene cultures. These data suggest that the development of obesity in the Zucker rat cannot be attributed to elevated hepatic lipogenesis in the fetus.

Chapter 22 Methodology and Utility of Primary Cultures of Hepatocytes from Experimental Animals

The chapter describes the methods employed for the culture of fetal, neonatal, and adult hepatocytes and cell lines derived from liver, and points out some of the experimental systems to which such cultures have been applied. A major advantage of primary adult liver cell cultures is the ability to maintain these cells in serum-free medium of known molecular composition, however there is a problem of the inability of the cells to continue through the cell cycle from G 2 into the mitotic phase. The chapter summarizes some of the biochemical approaches that are used to study primary cultures of mammalian hepatocytes. These include studies on intermediary metabolism and nucleic acid synthesis, protein secretion by cultures of hepatocytes, and drug and carcinogen metabolism by primary cultures of hepatocytes. One of the most critical areas in which hepatocytes are useful is the study of xenobiotic metabolism. The use of hepatic cell cultures in monitoring environmental carcinogens is potentially great, as most known chemical carcinogens are metabolized to active forms in the liver.

Time Dependence of the Glycogenic Effect of Insulin in Cultured Fetal Hepatocytes

Primary cultures of rat fetal hepatocytes were used to study the effect of insulin on glycogenesis during 24 h. Measurements of both glycogen content and 14C-glucose incorporation into glycogen gave similar results. A clear stimulatory insulin effect was observed within one hour, reaching a maximum after three hours. However, from 4 to 12 h, the effect declined strongly and then disappeared. Thereafter, it reappeared but to a lesser extent. These striking variations were not caused by a lack of active insulin in the medium or by any amplification of time variations in hepatocyte basal glycogenic capacity. They were induced by the hormone itself as a function of time of insulin presence at the hepatocyte level. Therefore, the effect of insulin is time dependent. The transient cessation in the stimulatory effect of insulin on net glycogenesis limits the total amount of glycogen stored and permits a more progressive glycogen accumulation. It is not yet clear whether this cessation is a result of a decrease in the insulin-stimulated glycogen synthesis, or an increase of glycogen breakdown, or both; but we showed that it does not require prior maximal stimulation of glycogenesis. After four hours of incubation with varying doses of insulin, a dosedependent decrease in the insulin glycogenic effect of a second maximal dose of hormone was observed, whereas glucagon-induced glycogenolysis was unchanged. The characteristics of the time dependence of the insulin effect suggest that it may play a regulatory role in fetal hepatocytes.

Hormonal Control and Putative Cell Cycle Dependency of AFP Production: Further Observations in vivo and in vitro

We describe the effect of hormones on AFP production in neonatal rats, in primary fetal rat hepatocyte cultures, in adult animals with liver injuries, and in tumor cell lines established in vitro. Preliminary data of studies on the putative cell cycle dependency of AFP production are also presented. Suppression of AFP by glucocorticoid hormones emerges as a most powerful system to study liver cell differentiation through AFP regulation.

Organ cultures of human fetal hepatocytes in the study of extra- and intracellular α1-antitrypsin

The rate of synthesis of α 1-antitrypsin has been studied in organ cultures of fetal human liver. By de novo synthesis, α 1-antitrypsin of the same electrophoretic mobility and molecular size as plasma α 1-antitrypsin was produced. Synthetic rate was comparable to in vivo conditions and was suppressed by cycloheximide, colchicine and neuraminidase. By increasing α 1-antitypsin levels in culture medium, suppression of α 1-antitrypsin release from the intra-to the extracellular site was achieved, i.e., synthesis does not proceed autonomously. This suppression was preceded by a temporary enhancement of synthesis. Both effects were found to be independent of degree of sialylation of added α 1-antitrypsin. In contrast to α 1-antitrypsinreleased in tissue culture, the intracellular protein, as analyzed by crossed immunoelectrophoresis of Triton X-100 extracts from fetal liver, was found to occur partly as slowly moving peaks. Whether these peaks represent proforms or incompletely glycosylated precursors of export α 1-antitrypsin or complexes with proteases remains unsettled. A variety of other plasma proteins are released in organ cultures making the system suitable for study of factors regulating plasma protein synthesis.

Regulation of fetal liver erythropoiesis

The liver is the main erythropoietic tissue of the human fetus at midterm. The regulatory mechanisms involved in red cell formation should explain three main aspects of fetal erythropoiesis. First, the initiation of erythropoiesis during the formation of the embryonic liver from the hepatic endoderm. Second, the conservation of a high erythropoietic activity in the fetal liver for several weeks until achievement of a fully developed bone marrow. And third, the switch mechanism from fetal hemoglobin to adult hemoglobin. There is experimental evidence for the induction of red cell formation in cells of the hepatic mesenchyme due to the interaction of these cells with the liver parenchymal cells. The nature of the factors involved in this process remain to be investigated but it has been found that at least one of them may be erythropoietin, a protein actively synthesized in cultures of fetal hepatocytes. Erythropoietin has been found in the fetal serum of mammals but seems to play a role late in the erythropoietic development of the liver. Liver erythroid cells isolated from young fetuses (8–12 weeks of gestation) show a poor response towards erythropoietin when compared with cells isolated from older fetuses. This observation suggests the involvement of other factors different from erythropoietin at early gestation. One of these factors is testosterone which stimulates heme and hemoglobin synthesis in liver cells isolated from fetuses of 10–13 weeks of gestation. Another set of factors are the hormones which interact with cellular β-andrenergic receptors. These factors act to regulate red cell formation in cells of fetuses obtained at 8 and 11 weeks of gestation. The switch mechanism from fetal to adult hemoglobin remains one of the main unresolved problems of fetal erythropoiesis. Erythropoietin does not change the ratios of fetal to adult hemoglobin in cell cultures of human fetal liver. Testosterone and estradiol added to these cells in vitro can change the ratio of both hemoglobins but since the change is small these steroid hormones may play only a secondary role in the switch mechanism of globin chains. The fetal lamb seems to have a switch mechanism different from the human because experimental bleeding (to increase erythropoietin concentration) elevates the adult hemoglobin levels. It has been suggested that in the sheep the regulation of adult hemoglobin synthesis (α 2β 2) may be indirectly controlled by an imbalance of γ to α chain mRNA's, which may change from 2 to 1 at early gestation to a ratio of 1:1 after 100 days of gestation. If a similar mechanism is operative in man remains to be investigated.

Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. IX. Specific inhibition of DNA synthesis initiation by very low density lipoprotein and possible significance to the problem of liver regeneration.

Rat serum very low density lipoprotein (VLDL) inhibits initiation of DNA synthesis in fetal rat hepatocyte cultures; cells engaged in synthesizing DNA resist inhibition. VLDL action is specific and apparently blocks prereplicative protein synthesis. These and other results, from studies of altered blood VLDL levels and [3H] thymidine incorporation into isolated liver nuclei in 70% hepatectomized normal and mutant hyperlipoproteinemic rats, as well as from infusion studies with a "mitogenic" hormone solution, suggest that hepatic VLDL metabolism is linked to the suppression of hepatocyte proliferation.

Role of cortisol on the glycogenolytic effect of glucagon and on the glycogenic response to insulin in fetal hepatocyte culture.

The effects of insulin and glucagon on glycogen metabolism were studied in cultured fetal hepatocytes transplanted from 15-day-old fetuses. The effects of these hormones were examined just after transplantation, when the cells contained only minute amounts of glycogen, and during the 3 to 4 day culture period, when the hepatocytes were exposed to 10 muM cortisol and actively accumulated glycogen. At all stages of the culture, glucagon addition (10 nM) was followed by a rapid depletion of labeled glycogen, previously synthesized during a pulse labeling with [14C]glucose: this effect was mimicked by N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) (0.3 to 1 nM). Such a glycogenolytic effect of glucagon was observed even 6 hours after transplantation, i.e. at a time when cortisol was not present. In addition, glucagon clearly induced cyclic adenosine 3':5'-monosphosphate (cyclic AMP) accumulation in cells grown for 18 hours in the absence of cortisol. With cells grown for 3 days in the presence of cortisol, glucagon-dependent glycogenolysis was also obtained when cortisol was removed from the medium 20 hours before hormone addition. Thus the presence of cortisol is not necessary either to maintain a response to glucagon or for the onset of the glycogenolytic effect of glucagon. Insulin addition (10 nM) stimulated [14C]glucose incorporation into glycogen at all stages of the culture when grown in the presence of cortisol; no glycogenic response to insulin was observed 6 hours after transplantation where cortisol was not previously introduced. In addition, if the hepatocytes were grown in the presence of insulin alone (i.e. in the absence of cortisol) no significant storage of glycogen occurred. Maximal storage (or labeling) of glycogen was observed when hepatocytes were grown in the presence of both cortisol and insulin. The presence of cortisol was therefore necessary for the expression of the glycogenic effect of insulin. These data show that marked difference exist between the onset of developmental responses towards glucagon and insulin. The glucagon-dependent regulatory pathway should be present very early in fetal development and should not depend on cortisol. On the contrary, the onset of the insulin-dependent regulatory pathway seems to be induced during culture, and it is likely that this is caused by cortisol.

Development of glycogen storage ability under cortisol control in primary cultures of rat fetal hepatocytes

Cortisol has been shown to induce glycogen storage function in primary cultures of fetal hepatocytes. The method we describe provides a homogeneous population of hepatocytes by elimination of hematopoietic cells. Hepatocytes transplanted from 15-day-old fetuses were grown in the absence or presence of cortisol (10 −5 M) for periods of up to 4 days. In the presence of cortisol, after a lag period (24 hr), the glycogen content increased sharply, regardless of whether the medium was replaced or not. Incorporation of radioactivity from (U) 14C-glucose into glycogen paralleled glycogen accumulation, but the specific activity of the stored glycogen was lower than the final specific activity of the glucose in the medium. This result shows that free glucose is a good precursor of glycogen but not the only one. Data from chase and labeling experiments prove that the hormone acts on the synthetic pathway. If cortisol was removed the glycogen content dropped, suggesting that glycogen synthesis depends on the continuous presence of the hormone. The in vitro maturation of hepatocyte can be provoked by the hormone before the normal in vivo maturation stage of the onset of glycogen accumulation. Other studies of the same in vivo phenomenon have demonstrated that accumulation of glycogen in the liver prior to birth is corticosteroid dependent, but only an in vitro study could clearly show that the hormone acts at the cellular level.

Aryl Hydrocarbon Hydroxylase Induction in Mammalian Liver Cell Culture: I. STIMULATION OF ENZYME ACTIVITY IN NONHEPATIC CELLS AND IN HEPATIC CELLS BY PHENOBARBITAL, POLYCYCLIC HYDROCARBONS, AND 2,2-BIS(p-CHLOROPHENYL)-1,1,1-TRICHLOROETHANE

Abstract Aryl hydrocarbon hydroxylase activity is inducible in rat fetal hepatocytes in culture by phenobarbital, the insecticide, 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (p,p'-DDT), benz[a]anthracene, or 3-methylcholanthrene. Between the 2nd and 4th day of age of the primary cultures, the maximal net rate at which the hydroxylase activity accumulates is about the same when phenobarbital, benz[a]anthracene, or 3-methylcholanthrene is in the growth medium at optimal concentrations. The optimal inducing concentrations for each compound are 2.0 mm phenobarbital, 100 µm p,p'-DDT, 13 µm benz[a]anthracene, and 0.75 µm 3-methylcholanthrene. The kinetic data of the oxygenase induction and degradation are the same in liver cell cultures exposed to either phenobarbital or benz[a]anthracene; the time required for doubling the hydroxylase activity is about 3.0 hours, and the initial half-life of the induced enzyme system is about 10½ hours. An additive effect is obtained when either phenobarbital or p,p'-DDT is present with a polycyclic hydrocarbon in the medium, but not when the hepatocytes are treated with phenobarbital plus p,p'-DDT, or with the combination of benz[a]anthracene plus 3-methylcholanthrene. In fetal liver cells exposed to phenobarbital for 3 days, total hepatocellular RNA synthesis does not change significantly or may increase slightly, whereas in hepatocytes treated with benz[a]anthracene for 1 to 3 days, gross RNA synthesis decreases about 30%. There is a 15 to 20% decline in total protein synthesis in liver cell cultures treated with either phenobarbital or benz[a]anthracene for 1 to 3 days. The apparent Michaelis constants for the control hydroxylase and the enzyme system inducible by either phenobarbital or benz[a]anthracene are similar, ranging between 2 and 4 µm in the presence of the substrate, benzo[a]pyrene.