Iron accumulation, iron-mediated toxicity and altered levels of ferritin and transferrin receptor in cultured astrocytes during incubation with ferric ammonium citrate

The cellular uptake and storage of iron have to be tightly regulated in order to provide iron for essential cellular functions while preventing the iron-catalysed generation of reactive oxygen species (ROS). In contrast to cells in other organs, little is known about the regulation of iron metabolism in brain cells, particularly in astrocytes. To investigate the regulation of iron metabolism in astrocytes we have used primary astrocyte cultures from the brains of newborn rats. After application of ferric ammonium citrate (FAC), cultured astrocytes accumulated iron in a time- (0-48 h) and concentration-dependent (0.01-1 mm) manner. This accumulation was prevented if FAC was applied in combination with the iron-chelator deferoxamine (DFX). Application of FAC to astrocyte cultures caused a strong increase in the cellular content of the iron storage protein ferritin and a decrease in the amount of transferrin receptor (TfR), which is involved in the transferrin-mediated uptake of iron into cells. In contrast, application of DFX strongly increased the level of TfR. Both up-regulation of ferritin content by iron application and up-regulation of TfR content by DFX were prevented by the protein synthesis inhibitor cycloheximide (CHX). During incubation of astrocytes with FAC, a mild and transient increase in the extracellular activity of the cytosolic enzyme lactate dehydrogenase and in the concentration of intracellular ROS was observed. In contrast, prevention of protein synthesis by CHX during incubation with FAC resulted in significantly more cell loss and a persistent and intense increase in the production of intracellular ROS. These results demonstrate that both iron accumulation and deprivation modulate the synthesis of ferritin and TfR in astrocytes and that protein synthesis is required to prevent iron-mediated toxicity in astrocytes.     

Calcein as a fluorescent iron chemosensor for the determination of low molecular weight iron in biological fluids

The fluorescence quenching of calcein (CA) is not iron specific and results in a negative calibration curve. In the present study, deferoxamine (DFO), a strong iron chelator, was used to regenerate the fluorescence quenched by iron. Therefore, the differences in fluorescence reading of the same sample with or without addition of DFO are positively and specifically proportional to the amounts of iron. We found that the same iron species but different anions (e.g. ferric sulfate or ferric citrate) differed in CA fluorescence quenching, so did the same anions but different iron (e.g. ferrous or ferric sulfates). Excessive amounts of citrate competed with CA for iron and citrate could be removed by barium precipitation. After optimizing the experimental conditions, the sensitivity of the fluorescent CA assay is 0.02 M of iron, at least 10 times more sensitive than the colorimetric assays. Sera from 6 healthy subjects were tested for low molecular weight (LMW) chelator bound iron in the filtrates of 10 kDa nominal molecular weight limit (NMWL). The LMW iron was marginally detectable in the normal sera. However, increased levels of LMW iron were obtained at higher transferrin (Tf) saturation (1.64–2.54 M range at 80% Tf saturation, 2.77–3.15 M range at 100% Tf saturation and 3.09–3.39 M range at 120% Tf saturation). The application of the assay was further demonstrated in the filtrates of human liver HepG2 and human lung epithelial A549 cells treated with iron or iron-containing dusts.     

The application of laser microprobe mass analysis to the study of biological material

Laser microprobe mass analysis (LAMMA) is an investigational method which is a powerful tool for the identification and quantitation of various elements present in small volumes of tissue. LAMMA is highly sensitive and capable of rapidly detecting concentrations of 1–3 p.p.m. of most metallic elements, in precisely localized cellular compartments. In order to further assess its value, cultured skin fibroblasts and biopsy tissues from human subjects and experimental animals were probed by LAMMA, and the results were correlated with ultrastructural findings. Biopsy samples were obtained from patients suffering from Gaucher disease, and from patients and animals with pathologic iron or copper metabolism. No significant abnormalities were detected in the cultured fibroblasts from patients with Gaucher disease, in contrast to the iron content of tissue biopsy Gaucher cells, which was markedly increased, apparently as a consequence of erythrophagocytosis. Particularly intense iron-related peaks were found in liver cytosiderosis due to neonatal or genetic haemochromatosis, thalassaemia major and in animal models of iron overload. An additional finding was the presence of aluminium accumulation in siderosomes of different cells. In liver biopsy samples from human Wilson's disease and from rats with an inherited disorder causing copper toxicosis, copper-containing compounds were identified and localized, and their relative concentration was estimated by LAMMA. The present study showed that LAMMA is a valuable technique for the localization and estimation of relative abundance of trace elements in various tissues containing excessive amounts of metals.     

Improved estimation of DNA fragment lengths from Agarose gels

A simple, sensitive assay for prolylcarboxypeptidase (PCP) is described. It utilizes a radiolabeled substrate, benzyloxycarbonyl-l-prolyl-l-[³H]alanine, and the details of its synthesis are also reported here. The hydrolysis of the dipeptide substrate is linear with respect to time or protein concentration until 10% of the substrate has been cleaved. Kinetic analysis yielded a K_m of 4.7 mm. The assay can be used to measure PCP activity in small amounts of biological fluid, homogenized tissue or cultured cells. Measurements of PCP activity in various cultured human cells showed endothelial cells from umbilical veins to have the highest activity (1625 ± 151 nmol/mg/h) followed by endothelial cells from umbilical artery (1017 ± 46 nmol/mg/h), human foreskin fibroblasts (719 ± 39 nmol/mg/h), and pulmonary artery endothelial cells (352 nmol/mg/h).     

Retinol supplementation induces DNA damage and modulates iron turnover in rat Sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular DNA damage probably involving cellular iron accumulation. Retinol (7μM) significantly induced DNA single strands breaks, DNA fragmentation and production of 8-oxo-7, 8-dihydro-2′-deoxyguanosine in cultured Sertoli cells. In contrast, lower doses seemed not to induce single-strands break in this experimental model. The breaks in DNA were inhibited by an iron scavenger; and 7μM retinol treatment modulated iron turnover leading to iron accumulation, suggesting that iron ions were required for the retinol cellular effects. These findings suggest that retinol-induced DNA damage was associated with the modulation of iron turnover, and these characteristics could be responsible for the increased incidence of lung cancer associated with retinoids supplementation.     

Effects of carboxylic acids on the uptake of non-transferrin-bound iron by astrocytes

The concentrations of non-transferrin-bound iron are elevated in the brain during pathological conditions such as stroke and Alzheimer's disease. Astrocytes are specialised for sequestering this iron, however little is known about the mechanisms involved. Carboxylates, such as citrate, have been reported to facilitate iron uptake by intestinal cells. Citrate binds iron and limits its redox activity. The presence of high citrate concentrations in the interstitial fluid of the brain suggests that citrate may be an important ligand for iron transport by astrocytes. This study investigates whether iron accumulation by cultured rat astrocytes is facilitated by citrate or other carboxylates. Contrary to expectations, citrate, tartrate and malate were found to block iron accumulation in a concentration-dependent manner; α-ketoglutarate had limited effects, while fumarate, succinate and glutarate had no effect. This blockade was not due to an inhibition of ferric reductase activity. Instead, it appeared to be related to the capacity of these carboxylates to bind iron, since phosphate, which also binds iron, diminished the capacity of citrate, tartrate and malate to block the cellular accumulation of iron. These findings raise the possibility that citrate may have therapeutic potential in the management of neurodegenerative conditions that involve cellular iron overload.     

Separate pathways for cellular uptake of ferric and ferrous iron

Separate pathways for transport of nontransferrin ferric and ferrous iron into tissue cultured cells were demonstrated. Neither the ferric nor ferrous pathway was shared with either zinc or copper. Manganese shared the ferrous pathway but had no effect on cellular uptake of ferric iron. We postulate that ferric iron was transported into cells via beta(3)-integrin and mobilferrin (IMP), whereas ferrous iron uptake was facilitated by divalent metal transporter-1 (DMT-1; Nramp-2). These conclusions were documented by competitive inhibition studies, utilization of a beta(3)-integrin antibody that blocked uptake of ferric but not ferrous iron, development of an anti-DMT-1 antibody that blocked ferrous iron and manganese uptake but not ferric iron, transfection of DMT-1 DNA into tissue culture cells that showed enhanced uptake of ferrous iron and manganese but neither ferric iron nor zinc, hepatic metal concentrations in mk mice showing decreased iron and manganese but not zinc or copper, and data showing that the addition of reducing agents to tissue culture media altered iron binding to proteins of the IMP and DMT-1 pathways. Although these experiments show ferric and ferrous iron can enter cells via different pathways, they do not indicate which pathway is dominant in humans.     

Heme regulation of HeLa cell transferrin receptor number

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.     

Retinol supplementation induces DNA damage and modulates iron turnover in rat Sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular DNA damage probably involving cellular iron accumulation. Retinol (7μM) significantly induced DNA single strands breaks, DNA fragmentation and production of 8-oxo-7, 8-dihydro-2'-deoxyguanosine in cultured Sertoli cells. In contrast, lower doses seemed not to induce single-strands break in this experimental model. The breaks in DNA were inhibited by an iron scavenger; and 7μM retinol treatment modulated iron turnover leading to iron accumulation, suggesting that iron ions were required for the retinol cellular effects. These findings suggest that retinol-induced DNA damage was associated with the modulation of iron turnover, and these characteristics could be responsible for the increased incidence of lung cancer associated with retinoids supplementation.     

Determination of glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) activity by high-performance liquid chromatography and electrochemical detection

The tripeptide glutathione (gamma-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis. The GCL assay described here is based on high-performance liquid chromatography and exploits the electrochemically active nature of gamma-glutamylcysteine (gamma-GC), the product of GCL activity. This method allows for the direct detection of gamma-GC rather than relying on derivatization of the molecule or linked assays. The sensitivity of the assay is sufficient to allow for the measurement of GCL activity in cultured cells. The specific activity of GCL in rat primary culture astrocytes was 9.7 +/- 1.7 nmol gamma-GC synthesized/min/mg protein.     

Determination of δ-aminolevulinate dehydratase activity by a specific fluorometric coupled-enzyme assay

A coupled-enzyme assay for the specific and sensitive determination of δ-aminolevulinate dehydratase activity has been developed. The assay specifically measured picomole quantities of the product, porphobilinogen, by its enzymatic conversion to uroporphyrinogen I and the fluorometric detection of oxidized uroporphyrin I. The coupled-enzyme assay was linear with time and protein concentration and required less than 3 h for 20 individual determinations. Under the standard assay conditions, 10 to 100 pmol of uroporphyrin I was reliably measured, representing 0.085 to 0.850 nmol/h of δ-aminolevulinate dehydratase activity per assay. In addition, the fluorometric assay was more sensitive than either the standard or the semimicro colorimetric methods. The specificity, rapidity, and sensitivity of this new fluorometric method facilitates the reliable determination of low levels of aminolevulinate dehydratase activity in small amounts of crude tissue homogenates or in cultured cells.     

Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1–100 nM were rapidly iron-depleted when ferric citrate—but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.     

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.     

Decreased heme content and cessation of cell growth in cultured chick embryo fibroblasts in the presence of horse serum: Stimulation of heme synthesis and cell growth by iron

A new spectrofluorometric method for heme quantitation in cultured fibroblasts is described. The method includes: (1) heme extraction by methanol/sulfuric acid, (2) partial purification of heme by a microchromatographic method, and (3) treatment of the purified heme by oxalic acid followed by fluorometric quantitation. Using this method, heme concentration was determined in chick embryo fibroblasts cultured in a medium supplemented with either 7% fetal bovine serum (FBS) or 10% horse serum (HS). In the presence of FBS, cultured cells actively divided and cells contained 34–55 pmol heme/mg protein. In contrast, cultures maintained in HS proliferated at a slower rate and contained 23–25 pmol heme/mg protein. The addition of 40 μM FeSO₄ to cultures maintained in the presence of HS stimulated cell proliferation, and the cellular heme concentration increased to 37–51 pmol/ mg protein. These findings suggest that the cessation of growth in the presence of HS may be due to decreased heme content in the cells and that the stimulation of cell growth by iron is mediated by its stimulation of heme synthesis.     

Melanosome–iron interactions within retinal pigment epithelium‐derived cells

Melanosomes were recently shown to protect ARPE‐19 cells, a human retinal pigment epithelium (RPE) cell line, against oxidative stress induced by hydrogen peroxide. One postulated mechanism of antioxidant action of melanin is its ability to bind metal ions. The aim here was to determine whether melanosomes are competent to bind iron within living cells, exhibiting a property previously shown only in model systems. The outcomes indicate retention of prebound iron and accumulation of iron by granules after iron delivery to cells via the culture medium, as determined by both colorimetric and electron spin resonance analyses for bound‐to‐melanosome iron. Manipulation of iron content did not affect the pigment's ability to protect cells against H₂O₂, but the function of pigment granules within RPE cells should be extended beyond a role in light irradiation to include participation in iron homeostasis.     

Reduction enhances yields of nitric oxide trapping by iron-diethyldithiocarbamate complex in biological systems.

The mechanism of NO trapping by iron-diethylthiocarbamate complexes was investigated in cultured cells and animal and plant tissues. Contrary to common belief, the NO radicals are trapped by iron-diethylthiocarbamates not only in ferrous but in ferric state also in the biosystems. When DETC was excess over endogenous iron ligands like citrate, ferric DETC complexes were directly observed with EPR spectroscopy at g=4.3. This was the case when isolated spinach leaves, endothelial cultured cells were incubated in the medium with 2.5mM DETC or mouse liver was perfused with 100mM DETC solution. After trapping NO, the nitrosylated Fe-DETC adducts are mostly in diamagnetic ferric state, with only a minor fraction having been reduced to paramagnetic ferrous state by endogenous biological reductants. In actual in vivo trapping experiments with mice, the condition of excess DETC was not met. The substantial quantities of iron in animal tissues were bound to ligands other than DETC, in particular citrate. These non-DETC complexes appear as roughly equal mixtures of ferric and ferrous iron. The presence of NO favors the replacement of non-DETC ligands by DETC. In all biological systems considered here, the nitrosylated Fe-DETC adducts appear as mixture of diamagnetic and paramagnetic states. The diamagnetic ferric nitrosyl complexes may be reduced ex vivo to paramagnetic form by exogenous reductants like dithionite. The trapping yields are significantly enhanced upon exogenous reduction, as proven by NO trapping experiments in plants, cell cultures and mice.     

Rapid Assay for Microbially Reducible Ferric Iron in Aquatic Sediments

The availability of ferric iron for microbial reduction as directly determined by the activity of iron-reducing organisms was compared with its availability as determined by a newly developed chemical assay for microbially reducible iron. The chemical assay was based on the reduction of poorly crystalline ferric iron by hydroxylamine under acidic conditions. There was a strong correlation between the extent to which hydroxylamine could reduce various synthetic ferric iron forms and the susceptibility of the iron to microbial reduction in an enrichment culture of iron-reducing organisms. When sediments that contained hydroxylamine-reducible ferric iron were incubated under anaerobic conditions, ferrous iron accumulated as the concentration of hydroxylamine-reducible ferric iron declined over time. Ferrous iron production stopped as soon as the hydroxylamine-reducible ferric iron was depleted. In anaerobic incubations of reduced sediments that did not contain hydroxylamine-reducible ferric iron, there was no microbial iron reduction, even though the sediments contained high concentrations of oxalate-extractable ferric iron. A correspondence between the presence of hydroxylamine-reducible ferric iron and the extent of ferric iron reduction in anaerobic incubations was observed in sediments from an aquifer and in fresh- and brackish-water sediments from the Potomac River estuary. The assay is a significant improvement over previously described procedures for the determination of hydroxylamine-reducible ferric iron because it provides a correction for the high concentrations of solid ferrous iron which may also be extracted from sediments with acid. This is a rapid, simple technique to determine whether ferric iron is available for microbial reduction.     

A microassay for heme oxygenase activity using thin-layer chromatography.

A sensitive and facile assay for heme oxygenase (HO) has been developed. The basis of the assay is the detection of [14C]bilirubin formation in a coupled enzyme assay involving HO and biliverdin reductase actions, respectively. Separation of substrate from product is accomplished by thin-layer chromatography with subsequent quantitation by liquid scintillation counting of radioactive material present on chromatograms. As little as 20 micrograms of total cellular protein derived from cells growing in a standard 25-cm2 culture flask is sufficient for detection of HO enzyme activity using this assay. The reaction is inhibited by tin-protoporphyrin (10 microM final concentration), a specific inhibitor of HO. The linearity of the enzyme reaction with respect to incubation time and amount of protein used was established. Comparison of the new HO assay with a spectrophotometric assay was made, and good agreement of the results from both methods was found. The assay described here should facilitate measurements of this important heme-degrading enzyme in tissue culture studies and cases where limited amounts of material are available.     

胰岛素对小鼠原代肝细胞甘油三酯合成分解代谢的影响

目的：建立小鼠肝细胞体外培养的方法,研究不同浓度胰岛素对肝细胞甘油三酯合成代谢、分解代谢及甘油三酯含量的影响。方法：通过肝脏灌注和胶原酶消化分离小鼠肝细胞,密度梯度离心纯化后,进行体外培养。在0 nmol/L,50 nmol/L,100 nmol/L,200 nmol/L胰岛素存在的情况下培养,通过3H标记的甘油测定细胞内甘油三酯合成速率,使用3H标记的油酸预孵育,加入triacsin C抑制脂肪酸重酯化,追踪掺入3H的甘油三酯分解的速率。采用甘油三酯检测试剂盒,测定不同浓度胰岛素对细胞内甘油三酯含量的影响。结果：成功分离了小鼠原代肝细胞,存活率达90%。50 nmol/L胰岛素对细胞甘油三酯含量及甘油三酯合成分解速率影响较小。100 nmol/L胰岛素可显著增加甘油三酯合成速率,减低分解速率,使细胞内甘油三酯含量增加。200 nmo/L胰岛素反而降低甘油三酯合成速率,细胞内甘油三酯含量少于对照组（0 nmol/L）。结论：本研究成功建立了小鼠原代肝细胞分离培养的方法,使用3H标记物敏感的检测肝细胞内甘油三酯合成分解速率。研究发现,过高浓度的胰岛素反而抑制肝细胞甘油三酯的储积。