The effect of dexamethasone on transferrin secretion by cultured fetal rat hepatocytes

Cultured fetal rat hepatocytes derived from 12, 15 and 19-day gestation rats are capable of secreting transferrin. When dexamethasone is added to the medium an increased secretion rate is observed. The changes in secretion rates in control as well as dexamethasone-treated cells during culture have been shown to correlate with the level of mRNA coding for transferrin. Immunocytochemical experiments show that initially all hepatocytes contain transferrin which is localized in the lumina of the perinuclear space, rough endoplasmic reticulum and in the saccules and vesicles of the Golgi apparatus. During culture, particularly in control cells, the intensity of labelling varies from cell to cell. In addition, adjacent cells are observed to label more intensely in different intracellular organelles.     

Effects of magnesium and iron on lipid peroxidation in cultured hepatocytes

In primary cultures of rat hepatocytes, the effects of extracellular Mg²⁺ and Fe on lipid peroxidation (LPO) as measured by means of malondialdehyde (MDA) formation were investigated.Incubation of hepatocytes at decreasing extracellular Mg²⁺ concentration enhanced LPO, depending on extracellular Fe. About 96% of MDA accumulated in the culture medium. Addition of desferrioxamine prevented LPO.Additionally, the formation of oxygen free radicals was determined by fluorescence reduction of cis-parinaric acid. With this method, an immediate decay of fluorescence was found after addition of Fe²⁺. Fluorescence reduction was completely prevented by desferrioxamine, indicating the function of extracellular Fe. This mechanism may operate additionally to the increase in intracellular Fe and intracellular formation of oxygen free radicals during Mg deficiencyin vivo.     

Protection of human and murine hepatocytes against Fas-induced death by transferrin and iron

Recent studies in a murine model show that transferrin (Tf) interferes with Fas-mediated hepatocyte death and liver failure by decreasing pro-apoptotic and increasing anti-apoptotic signals. We show here in vitro in murine and human hepatocyte cell lines and in vivo in mice that Fas-induced apoptosis is modulated by exogenous Tf and iron. The results obtained with iron-free Tf (ApoTf), iron-saturated Tf (FeTf), and the iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in its iron-free and iron-saturated (FeSIH) forms indicate that apoptosis-modulating effects of Tf are not mediated by iron alone. Both the Tf molecule and iron affect multiple aspects of cell death, and the route of iron delivery to the cell may be critical for the final outcome of cellular Fas signaling. Survival of hepatocytes ‘stressed’ by Fas signals can be manipulated by Tf and iron and may be a target for prophylactic and therapeutic interventions.     

Effects of iron deficiency and iron overload on manganese uptake and deposition in the brain and other organs of the rat

Managanese (Mn) is an essential trace element at low concentrations, but at higher concentrations is neurotoxic. It has several chemical and biochemical properties similar to iron (Fe), and there is evidence of metabolic interaction between the two metals, particularly at the level of absorption from the intestine. The aim of this investigation was to determine whether Mn and Fe interact during the processes involved in uptake from the plasma by the brain and other organs of the rat. Dams were fed control (70 mg Fe/kg), Fe-deficient (5–10 mg Fe/kg), or Fe-loaded (20 g carbonyl Fe/kg) diets, with or without Mn-loaded drinking water (2 g Mn/L), from day 18–19 of pregnancy, and, after weaning the young rats, were continued on the same dietary regimens. Measurements of brain, liver, and kidney Mn and nonheme Fe levels, and the uptake of⁵⁴Mn and⁵⁹Fe from the plasma by these organs and the femurs, were made when the rats were aged 15 and 63 d. Organ nonheme Fe levels were much higher than Mn levels, and in the liver and kidney increased much more with Fe loading than did Mn levels with Mn loading. However, in the brain the increases were greater for Mn. Both Fe depletion and loading led to increased brain Mn concentrations in the 15-d/rats, while Fe loading also had this effect at 63 d. Mn loading did not have significant effects on the nonheme Fe concentrations.⁵⁴Mn, injected as MnCl₂ mixed with serum, was cleared more rapidly from the circulation than was⁵⁹Fe, injected in the form of diferric transferrin. In the 15-d-rats, the uptake of⁵⁴Mn by brain, liver, kidneys, and femurs was increased by Fe loading, but this was not seen in the 63-d rats. Mn supplementation led to increased⁵⁹Fe uptake by the brain, liver, and kidneys of the rats fed the control and Fe-deficient diets, but not in the Fe-loaded rats. It is concluded that Mn and Fe interact during transfer from the plasma to the brain and other organs and that this interaction is synergistic rather than competitive in nature. Hence, excessive intake of Fe plus Mn may accentuate the risk of tissue damage caused by one metal alone, particularly in the brain.     

The effect of the flavonol rutin on serum and liver iron content in a genetic mouse model of iron overload

The flavonol rutin has been shown to possess antioxidant and iron chelating properties in vitro and in vivo. These dual properties are beneficial as therapeutic options to reduce iron accumulation and the generation of reactive oxygen species (ROS) resultant from excess free iron. The effect of rutin on iron metabolism has been limited to studies performed in wildtype mice either injected or fed high-iron diets. The effect of rutin on iron overload caused by genetic dysregulation of iron homoeostasis has not yet been investigated. In the present study we examined the effect of rutin treatment on tissue iron loading in a genetic mouse model of iron overload, which mirrors the iron loading associated with Type 3 hereditary haemochromatosis patients who have a defect in Transferrin Receptor 2 (TFR2). Male TFR2 knockout (KO) mice were administered rutin via oral gavage for 21 continuous days. Following treatment, iron levels in serum, liver, duodenum and spleen were assessed. In addition, hepatic ferritin protein levels were determined by Western blotting, and expression of iron homoeostasis genes by quantitative real-time PCR. Rutin treatment resulted in a significant reduction in hepatic ferritin protein expression and serum transferrin saturation. In addition, trends towards decreased iron levels in the liver and serum, and increased serum unsaturated iron binding capacity were observed. This is the first study to explore the utility of rutin as a potential iron chelator and therapeutic in an animal model of genetic iron overload.     

Role of transferrin in iron transport between maternal and fetal circulations of a perfused lobule of human placenta

The transfer of iron between the maternal and fetal circulations of an isolated perfused lobule of term human placenta was investigated using 125I-labelled or 59Fe-labelled diferric transferrin. There was negligible transplacental transfer of intact transferrin whereas nearly 4 per cent of the added 59Fe was transferred into the fetal circulation after 2 h, where it became associated with fetal transferrin. Over 20 per cent of the added 59Fe radioactivity was sequestered within the placental tissue during this period, associated with transferrin, ferritin and other uncharacterized molecules. This suggests an important role for an intracellular pool in regulating transfer. The presence of 10 mM chloroquine in the maternal circulation substantially reduced tissue accumulation of 59Fe and totally inhibited transfer to the fetus. It is concluded that the initial stages of iron transfer to the fetus involve the internalization of maternal iron-saturated transferrin bound to membrane receptors by receptor-mediated endocytosis, which can be inhibited by the drug chloroquine. Subsequently, the transplacental transfer of iron to the fetus does not involve the concomitant movement of transferrin.     

Triiodothyronine receptor sites in serum-free cultured hepatocytes from adult rat liver

Nuclear T3 specific binding sites were characterized by Scatchard analyses of L-125I-T3 binding to nuclei extracted from freshly isolated and 1, 2 and 6 day-cultured hepatocytes. The results demonstrate a marked decrease in T3 binding capacity of nuclei extracted from 1 day-cultured cells followed by an almost complete recovery within 6 days. The affinity constant value of nuclear receptor sites is significantly decreased in 1 day-cultured cells with a subsequent partial recovery. The affinity and capacity pattern of nuclear T3 binding sites appears to be in line with the delayed responses of hepatocyte primary cultures to T3.     

Effect of D-Penicillamine on iron uptake by isolated rat hepatocytes

d-penicillamine (β,β-dimethylcysteine) promotes the incorporation of iron into isolated rat hepatocytes. The mechanism for doing this remains unknown. No differences in iron distribution between control and treated cells has been observed. Ferritin appears as the main destination of internalized iron in both cases. Therefore, increasing iron storage may appear as a side effect of the use ofd-penicillamine as a therapeutic agent for several diseases.     

Effect of corynetoxin isolated from parasitized annual ryegrass on albumin and transferrin synthesis and secretion by cultured fetal rat hepatocytes

A group of glycolipid toxins, corynetoxin (CT), isolated from parasitized annual ryegrass, was shown to suppress the synthesis of both albumin and transferrin by cultured fetal rat hepatocytes. Based on [³H]leucine incorporation, inhibition of transferrin synthesis was greater than that of both albumin and total protein synthesis. As a result, the secretion of albumin and transferrin was decreased. The incorporation of [³H]N-AcGlc into cellular glycoproteins was only marginally affected by CT, although a dramatic reduction was observed with respect to the secreted proteins. Transferrin secreted into the culture medium was substantially non-glycosylated, judging by the absence of [³H]N-AcGlc. These studies suggested that the toxin preferentially affects the synthesis, and hence the secretion of glycoproteins, although it did not block the secretion of the proteins albumin and transferrin, as these did not accumulate intercellularly. Since transferrin labelled with [³H]leucine but not [³H]N-AcGlc is detected in the culture medium of hepatocytes exposed to CT, it was concluded that glycosylation of the protein is not required for secretion. This study shows that the effects of CT on protein synthesis and secretion in cultured hepatocytes are similar to those reported for tunicamycin (TM).     

The effect of lead on iron uptake from transferrin in human erythroleukemia (K562) cells

The effect of lead on cellular iron metabolism has been investigated using human erythroleukemia (K562) cells. When the cells were cultured with 100 m Pb²⁺ for 48 h, the rate of cellular iron uptake from transferrin decreased to 46% of that in untreated cells. Scatchard analysis of the binding data revealed that this reduction was the result of a decrease in the number of transferrin receptors rather than an alteration in ligand-receptor affinity. The results of immunoprecipitation of transferrin receptors on the cell surface also confirmed the decreased expression of transferrin receptors by lead-treated cells. The down-regulation of transferrin receptors by treatment with lead did not result from a decrease in the total amount of the receptor, as determined by immunoblotting. Moreover, the biosynthesis of the receptor was unaffected by lead treatment. Thus, the down-regulation of surface transferrin receptors in lead-treated cells might be due to a redistribution of receptors rather than an actual loss of receptors from the cell. Using kinetic analysis, it was shown that redistribution of the receptor did not result from the alteration in the rates of transferrin receptor recycling. A comparison of the amounts of transferrin receptor on the cell surface and in the cycling pool revealed that the sequestration of the receptor from normal flow through the cycle might cause down-regulation of the surface receptor.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.     

Adult rat hepatocytes cultured on an oxygen-permeable film increases the activity of albumin secretion

Primary culture of rat hepatocyte was performed in an oxygen-permeable film dish (F-dish), which would be expected to give an oxygen-rich culture condition. In the conventional culture dish in which the depth of medium was 2 mm, the oxygen tension (pO₂) in the medium decreased from 19% (144 mmHg) to 0.3% (2.3 mmHg) within 2 hr, while the pO₂ in the F-dish maintained 8.5% (64.6 mmHg) even after 2 hr. The adverse effect of the oxygen-deficiency appeared in the albumin secretion activity of the hepatocytes and it was more remarkable in the early period of culture. The average rate of albumin secretion for the initial 48 hr was 2.0 μg ml^-1 hr^-1 or 96 μg 10⁶ cells^-1 day^-1 in the F-dish. The average rate of albumin secretion for the initial 12 hr was only 0.36 μg ml^-1 hr^-1 in the conventional culture dish. The activity of ammonia elimination in the F-dish was 20–50% higher than the conventional culture dish. Three-dimensional aggregate was formed only in the F-dish. The advantage of three-dimensional aggregate for albumin secretion was not clear compared with two-dimensional monolayer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Involvement of transferrin in the reduction of iron by the transplasma membrane electron transport system

Nonpermeable electron acceptors can be reduced by a transplasma membrane electron transport system in suspensions of intact cells. Here we report that diferric transferrin is reduced by HeLa S3 cells. The reduction is recorded spectrophotometrically as the formation of the ferrous complex of bathophenanthroline disulfonate. Ferric ammonium citrate can also be used as an electron acceptor, and the presence of low concentrations of diferric transferrin greatly stimulates the reduction of trivalent iron under these conditions. Likewise very low concentrations of ferricyanide, which does not give rise to a ferrous bathophenanthroline disulfonate complex formation, have a strong stimulatory effect on the complex formation when ferric ammonium citrate is the source of ferric iron. Apotransferrin is a potent inhibitor of the reaction. The inhibition occurs at the concentration necessary for complete occupancy of the transferrin receptors. The inhibition can be demonstrated also when high concentrations of ferricyanide are used as electron acceptor. The possible mechanism behind the reported phenomena is discussed, and it is concluded that the transplasma membrane electron transport system can be involved in the process of cellular iron uptake.     

Intermediate steps in cellular iron uptake from transferrin. II. A cytoplasmic pool of iron is released from cultured cells via temperature-dependent mechanical wounding

Summary A previous study described a cytoplasmic, transferrin (Tf)-free, iron (Fe) pool that was detected only when cells were mechanically detached from the culture substratum at 4°C, after initial incubation with⁵⁹Fe-¹²⁵I-Tf at 37°C (Richardson and Baker, 1992a). The release of this internalized⁵⁹Fe could be markedly reduced if the cells were treated with proteases or incubated at 37°C prior to detachment. The present study was designed to characterize this Fe pool and understand the mechanism of its release. The results show that cellular⁵⁹Fe release increased linearly as a function of preincubation time with⁵⁹Fe-Tf subsequent to mechanical detachment at 4°C using a spatula. These data suggest that the⁵⁹Fe released was largely composed of end product(s) and was not an “intermediate Fe pool.” When the Fe(II) chelator, dipyridyl (DP), was incubated with⁵⁹Fe-Tf and the cells, it prevented the accumulation of⁵⁹Fe that was released following mechanical detachment at 4°C. Other chelators had much less effect on the proportion of⁵⁹Fe released. Examination of the⁵⁹Fe released showed that after a 4-h preincubation with⁵⁹Fe-Tf, approximately 50% of the⁵⁹Fe was present in ferritin. These data indicate that mechanical detachment of cells at 4°C resulted in membrane disruptions that allow the release of high M, molecules. Moreover, electron microscopy studies showed that detachment of cells from the substratum at 4°C resulted in pronounced membrane damage. In contrast, when cells were detached at 37°C, or at 4°C after treatment with pronase, membrane damage was minimal or not apparent. These results may imply that temperature-dependent processes prevent the release of intracellular contents on membrane wounding, or alternatively, prevent wounding at 37°C. The evidence also indicates that caution is required when interpreting data from expriments where cells have been mechanically detached at 4°C.     

Chronic iron overload inhibits protein secretion by adult rat hepatocytes maintained in long-term primary culture

Short-term pure cultures and long-term cocultures of adult rat hepatocytes with rat liver epithelial cells, presumably derived from primitive biliary cells, were used to define in vitro models of iron overloaded hepatocytes in order to understand the molecular mechanism responsible for liver damage occurring in patients with hemochromatosis. In vitro iron overload was obtained by daily addition of ferric nitrilotriacetate to the culture medium. A concentration of 20 microM ferric salt induced hepatocyte iron overload with minimal cytotoxicity as evaluated by cell viability, morphological changes of treated cells and cytosolic enzyme leakage into the culture medium. The effects of iron overload on protein biosynthesis and secretion were studied in both short-term pure cultures and long-term cocultures of hepatocytes. The amounts of intracellular and newly synthesized proteins were never modified by the iron treatment. Furthermore, neither the relative amounts of transferrin and albumin mRNAs nor their translational products were altered by iron overload. Moreover, no change in the transferrin isomeric forms were observed in treated cells. In contrast, a prolonged exposure of cocultured hepatocytes to 20 microM ferric salt led to a significant decrease in the amount of proteins secreted in the medium. This decrease included the two major secreted proteins, namely albumin and transferrin, and probably all other secreted proteins. These results demonstrate that iron loading alters neither the total nor the liver specific protein synthesis activity of cultured hepatocytes. They suggest that chronic overload may impede the protein secretion process.     

Persistent biologic actions of insulin on cultured fetal human fibroblasts

Summary Monolayer cultures of fetal human fibroblasts, preincubated in serum-free culture medium overnight (about 18 hr), were incubated with insulin (0.1 to 100 mU per ml), then washed and incubated in an insulin-free medium. The effect of insulin on glucose utilization, uridine incorporation into RNA and leucine incorporation into protein was maintained after removal of insulin and washing. For both glucose utilization and uridine incorporation into RNA, this effect was demonstrated at physiologic levels of insulin (0.1 mU per ml). When anti-insulin serum was added to the cultures after the cell preincubated with insulin were washed, this effect was greatly attenuated. This lasting effect of insulin was probably not due to nonspecifically bound insulin becoming available to the cells. Binding of¹²⁵I-monoiodoinsulin was examined in monolayer cultures of fetal human fibroblasts. When unlabeled insulin was present at about 1 mU per ml concentration, 50% displacement of monoiodoinsulin occured. When fibroblasts were incubated with monoiodoinsulin and then removed from the radioactive medium, initial dissociation of the bound hormone occurred rapidly but then reached a plateau. This prolonged insulin effect appears to result from persistent binding of insulin to its receptor. Supported in part by PHS Grants AM-02456, AM-05020 and AM-15312, by the Kroc Foundation and by the Diabetes Center (AM-17047). Supported in part by Research Career Development Award AM-47142 from NIAMDD.     

Effects of ferrous iron and transferrin on cell proliferation of human diploid fibroblasts in serum-free culture

Summary Iron-free RITC 80-7 defined medium was used to examine effects of ferrous iron and transferrin on cell proliferation of human diploid fibroblasts. Both ferrous iron and holotransferrin stimulated cell proliferation in the medium, but apotransferrin did not. When 5 g/l human serum albumin (HSA) was added to the defined medium, excellent growth was obtained under hypoxic conditions, whereas a reduction of cellular growth during the culture periods was observed under aerobic conditions. When ferrous iron was added to the HSA medium alone, the reduction in growth increased in proportion to the concentrations, whereas the addition of transferrin prevented this reduction in a concentration-dependent manner. This suggests that the ferrous iron concentration in media causes a reduction in growth under aerobic conditions and transferrin prevents this reduction because it decreases the ferrous iron concentration. Further, serum albumin seems to be a source of iron in media.     

Increase in cellular pool of low-molecular-weight iron during ethanol metabolism in rat hepatocyte cultures

Ethanol-induced lipid peroxidation was studied in primary rat hepatocyte cultures supplemented with ethanol at the concentration of 50 mM. Lipid peroxidation was assessed by two indices: (1) conjugated dienes by second-derivative UV spectroscopy in lipid extract of hepatocytes (intracellular content), and (2) free malondialdehyde (MDA) by HPLC-UV detection and quantitation for the incubation medium (extracellular content). In cultures supplemented with ethanol, free MDA increased significantly in culture media, whereas no elevation of conjugated diene level was observed in the corresponding hepatocytes. The cellular pool of low-mol-wt (LMW) iron was also evaluated in the hepatocytes using an electron spin resonance procedure. An early increase of intracellular LMW iron (≤1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas α-tocopherol, which prevented lipid peroxidation, did not inhibit the increase of LMW iron. Therefore, the LMW iron elevation was the result of ethanol metabolism and was not secondarily induced by lipid hydroperoxides. Thus, ethanol caused lipid peroxidation in rat hepatocytes as shown by the increase of free MDA, although no conjugated diene elevation was detected. During ethanol metabolism, an increase in cellular LMW iron was observed that could enhance conjugated diene degradation.     

Cadmium-induced hepatotoxicity in cultured rat hepatocytes as evaluated by morphometric analysis

Summary Freshly isolated hepatocytes from neonatal rats were cultured for approximately 24 h; incubated for 5, 30, or 60 min in solutions containing 0, 50, 100, or 200 μM cadmium; embedded in plastic; and sectioned for optical microscopy. The exeent of cadmium-induced hepatotoxicity was evaluated by double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of threedimensional information) whereby hepatocytes were classified on the basis of the severity of morphologic damage at the optical level. Both time and concentration effects were studied. Cultures exposed to 200 μM cadmium, for various intervals of time from 5 to 60 min, showed statistically significant reductions in the relative volume percent of normal hepatocytes, elevations (then reductions) in the relative volume percent of slightly damaged hepatocytes, increases in the relative volume percent of moderately damaged cells, and increases in the relative volume percent of severely damaged liver cells. As the concentration of cadmium was increased from 50 to 200 μM cadmium (during both 30 and 60-min exposures), significant trends were observed in cellular distribution patterns based on relative volume percent. Morphologically normal cells decreased, both slightly damaged and moderately damaged cells increased, and severely damaged cells remained unchanged. These results indicated that morphometric analysis at the optical level provided quantitative estimates for the evaluation of time- and concentration-effects of cadmium on cultured hepatocytes. This work was supported by a grant from the Johns Hopkins University Center for Alternatives to Animal Testing.     

Influence of transferrin glycans on receptor binding and iron-donation

Human bi-bi-antennary transferrin (Tf) was partially deglycosylated by subsequently incubating with one or more of the following exoglycosidases: neuraminidase, β-galactosidase or N-Acetyl-β-D-glucosaminidase. Aglyco-Tf obtained from serum of a patient suffering from the Carbohydrate Deficient Glycoprotein syndrome was isolated. Receptor binding and the Tf and iron uptake capacities of the fully glycosylated-, partially deglycosylated- and aglyco-Tf were compared using the human hepatoma cell line PLC/PRF/5. No difference in binding capacity between the iso-Tf fractions could be demonstrated, however, the Tf and iron uptake capacity of aglyco-Tf was clearly reduced compared with the other Tf fractions. This revised version was published online in November 2006 with corrections to the Cover Date.