Selenium deficiency in cultured adrenocortical cells: restoration of glutathione peroxidase and resistance to hydroperoxides on addition of selenium

Cultured bovine adrenocortical cells were previously shown to be functionally deficient in selenium and vitamin E when grown in medium supplemented with fetal bovine serum. In the present experiments, the lack of significant bioavailable amounts of selenium in the medium was demonstrated by the finding of only low levels of glutathione peroxidase in the cultured cells (0.008 U/mg protein compared with 0.045 U/mg protein in fresh adrenocortical tissue). When 20 nM selenium as selenite was added to the cultured adrenocortical cells, glutathione peroxidase activity increased continuously over 72 h, with a total increase of about eightfold over this period. Over the same time-course, the highest concentration of cumene hydroperoxide tolerated by the cells without cell death increased progressively from 10 microM to 50 microM. Addition of 1 microM alpha-tocopherol also increased the amount of cumene hydroperoxide tolerated to 50 microM. Cell death was measured by cloning efficiency after removal of cumene hydroperoxide. Addition of either selenium or alpha-tocopherol had little effect on the growth rate of the cells over six passages, even when residual vitamin E was removed from the serum by extraction with ether and residual low molecular weight selenium compounds were removed by dialysis. It is concluded that combined deficiency of selenium and vitamin E, at least in the presence of other components of fetal bovine serum, has little effect on the ability of the cells to survive under normal conditions, as evidenced by continued long-term proliferation. However, the low levels of glutathione peroxidase resulting from selenium deficiency cause an increase susceptibility to peroxide-mediated toxicity. The combined deficiency of selenium and vitamin E impairs the ability of cells to survive under adverse conditions, as well as altering mitochondrial functions, as previously demonstrated.     

Effect of increasing selenite concentrations, vitamin E supplementation and different fetal calf serum content on GPx1 activity in primary cultured rabbit hepatocytes

Primary rabbit hepatocytes from 6 week old female New Zealand White rabbits (3.0 x 10(6) viable hepatocytes per treatment) were incubated for 24 h or 48 h with two basic variants of the selenium and vitamin E free DMEM/F12-HAM nutrition medium containing 2.5% or 10% fetal calf serum (FCS). Selenium and vitamin E concentrations of the media were varied by the addition of 0, 10, 50 and 100 ng Se/mL medium as sodium selenite and 100 microg alpha-tocopheryl acetate/mL. Lactic dehydrogenase (LDH) leakage of the hepatocytes was not influenced by the various selenium concentrations of the media, whereas vitamin E addition significantly inhibited LDH release. The activity of cellular glutathione peroxidase (GPx1) was markedly induced by increasing the selenium supplementation of the culture media. Vitamin E supply further enhanced GPx1 induction. In hepatocytes cultivated at the lower serum concentration (2.5% FCS), increasing the selenite concentration of the media raised GPx1 and reduced the intracellular levels of the reduced tripeptide glutathione (GSH). No vectored relation between the selenium concentration of the media and the activity of superoxide dismutase (SOD) could be observed. After both incubation periods (24 h and 48 h) SOD activity was significantly higher in the cytosol of hepatocytes grown in media containing 10% FCS as compared to cells incubated at the 2.5% FCS level. Furthermore, SOD activity was reduced by the addition of vitamin E to the media. In conclusion the results indicate an effective metabolism of rabbit hepatocytes for selenite even in amounts as low as nanograms. A general cytoprotective role for vitamin E can be shown by its ability to decrease LDH leakage and by the reduction of SOD activity.     

The importance of selenium in the prenatal and postnatal development of calves and lambs.

Selenium deficiency is responsible for Zenker type muscle degeneration in calves, lambs, and foals in the prenatal and postnatal stages of development. Investigations have shown that the selenium GSH Px, and vitamin E content of the maternal and fetal parts of the placenta in cattle are different. Similarly, low concentrations of selenium are present in milk from cows and sheep. In addition to an inadequate supply of selenium and vitamin E as a contributory cause of fetal nutritive muscular dystrophy (FNMD), it is assumed that a placental transport block and/or impaired selenium metabolism in the placenta are also responsible. Postnatal nutritive muscular dystrophy, however, is attributed to either acute selenium and vitamin E deficiency in basic feed or impaired plant absorption of selenium as a result of antagonistic elements, such as sulphur.     

The importance of selenium in the prenatal and postnatal development of calves and lambs

Selenium deficiency is responsible for Zenker type muscle degeneration in calves, lambs, and foals in the prenatal and postnatal stages of development. Investigations have shown that the selenium GSH P_x, and vitamin E content of the maternal and fetal parts of the placenta in cattle are different. Similarly, low concentrations of selenium are present in milk from cows and sheep. In addition to an inadquate supply of selenium and vitamin E as a contributory cause of fetal nutritive muscular dystrophy (FNMD), it is assumed that a placental transport block and/or impaired selenium metabolism in the placenta are also responsible. Postnatal nutritive muscular dystrophy, however, is attributed to either acute selenium and vitamin E deficiency in basic feed or impaired plant absorption of selenium as a result of antagonistic elements, such as sulphur.     

Optimization of Conditions for Production of Channel Catfish Ovary Cells and Channel Catfish Virus DNA

Medium supplements were examined for their effect on the growth of channel catfish ovary cells. It was found that the usual serum supplement of 10% fetal calf serum could be successfully replaced with a combination of 5% fetal calf serum and a mixture of insulin, transferrin, and selenous acid. It was also found that these cells could be grown in a more efficient manner on microcarrier beads. This type of culture produced 14 times the number of cells per milliliter of total medium used compared with the usual tissue culture flasks used for cell growth. The microcarrier system also provided for greater production efficiency of DNA from channel catfish virus, a virus that infects this cell line.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

Combined Therapeutic Effect of Dietary Selenium and Vitamin Ε on Manifested Vesd Syndrome in Weaned Pigs

By using a therapeutic dietary supplementation in pigs, which had developed the vitamin Ε and selenium deficiency (VESD) syndrome, the same amounts of α-tocopheryl acetate and selenium were found to be effective as under prophylactic conditions. The experiment thus supported the conclusions that the addition of 5 mg DL-α-tocopheryl acetate/kg and 135 μg selenium/kg to a diet, which contained only traces of vitamin Ε and selenium, represents a level of minimal requirement. Glutathione peroxidase activity in blood serum was used to evaluate the selenium status in pigs. A modified method for determination of tocopherol in fat tissue was described. The addition of 15 mg α-tocopheryl acetate/kg diet was demonstrated to be sufficient to maintain the tocopherol stores in body fat at an unchanged level.     

Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation in vitro.

We have compared several methods for reducing calcium and magnesium concentrations in tissue culture medium, with the objective of producing selective deficiency effects on the growth of mouse (L5178Y) and human (P1R) lymphoblasts. In experiments in which calcium- and magnesium- "free" McCoy's medium was supplemented with 15% horse or fetal calf serum, enough calcium and magnesium was provided by serum to support normal lymphoblast growth rate. Either dialysis or chelating-resin treatment of horse or fetal calf serum reduced calcium and magnesium contents approximately 100-fold. Use of dialyzed sera resulted in reduced growth rate, although in most cases the reduction in growth could be attributed to other effects of dialysis on serum, inasmuch as growth in those experiments was not restored to normal by the addition of calcium and magnesium to the medium. In contrast, the reduction of lymphoblast growth rate that occurred when resin-treated serum was used was always attributable to removal of calcium and magnesium, as normal growth always occurred in cultures to which calcium and magnesium were added. To demostrate a growth-inhibiting effect on either mouse or human lymphoblasts by severe reduction of either calcium or magnesium in the presence of normal amounts of the alternative cation, it was necessary to (a) expose McCoy's Ca-Mg-"free" medium to chelating-resin to reduce further the residual cation concentrations; (b) wash cells from stock cultures in a medium devoid of calcium and magnesium prior to inoculation into experimental cultures; (c) reduce the proportion of serum in the final medium from 15 to 5%; and (d) add 100 muM EGTA to cultures. Under these conditions, growth of both cell types was completely abolished in the presence of normal magnesium but in the absence of added calcium, and markedly reduced in the presence of normal calcium but in the absence of magnesium. These modifications did not compromise growth in cultures containing normal concentrations of both ions.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Growth of Myeloma MPC-11 Cells in Serum-free Growth Factor Supplemented Medium

A myeloma MPC-11 cell line, which originated in a mouse plasma cell tumor, was able to proliferate in the absence of serum in a synthetic medium supplemented with transferrin, ethanolamine and selenium. The cells showed almost the same growth rate in this medium as in medium with 10% fetal calf serum added. Immunoglobulins secreted into the serum-free medium by the cells were easily separated from medium components by polyacrylamide gel electrophoresis.     

Cell death caused by selenium deficiency and protective effect of antioxidants

Selenium is an essential trace element and it is well known that selenium is necessary for cell culture. However, the mechanism underlying the role of selenium in cellular proliferation and survival is still unknown. The present study using Jurkat cells showed that selenium deficiency in a serum-free medium decreased the selenium-dependent enzyme activity (glutathione peroxidases and thioredoxin reductase) within cells and cell viability. To understand the mechanism of this effect of selenium, we examined the effect of other antioxidants, which act by different mechanisms. Vitamin E, a lipid-soluble radical-scavenging antioxidant, completely blocked selenium deficiency-induced cell death, although alpha-tocopherol (biologically the most active form of vitamin E) could not preserve selenium-dependent enzyme activity. Other antioxidants, such as different isoforms and derivatives of vitamin E, BO-653 and deferoxamine mesylate, also exerted an inhibitory effect. However, the water-soluble antioxidants, such as ascorbic acid, N-acetyl cysteine, and glutathione, displayed no such effect. Dichlorodihydrofluorescein (DCF) assay revealed that cellular reactive oxygen species (ROS) increased before cell death, and sodium selenite and alpha-tocopherol inhibited ROS increase in a dose-dependent manner. The generation of lipid hydroperoxides was observed by fluorescence probe diphenyl-1-pyrenylphosphine (DPPP) and HPLC chemiluminescence only in selenium-deficient cells. These results suggest that the ROS, especially lipid hydroperoxides, are involved in the cell death caused by selenium deficiency and that selenium and vitamin E cooperate in the defense against oxidative stress upon cells by detoxifying and inhibiting the formation of lipid hydroperoxides.     

Mitogenic effect of estradiol on MCF-7 human breast cancer cells can be modulated by serum

The MCF-7 human breast cancer cell line responds to estradiol stimulation in vitro by increased proliferation only if prolonged subcultures in dextran-coated charcoal-treated fetal calf serum have been made previously. This growth stimulation is not obtained when cells are grown in medium containing 5% untreated fetal calf serum. We describe here the culture conditions under which we obtain a reproducible estradiol effect on cell growth.     

Effect of selenium and vitamin E dietary deficiencies on chick lymphoid organ development

Diets specifically deficient in selenium (Se) and/or vitamin E or adequate in both nutrients were fed to chicks from the time of hatching. Lymphoid organs (bursa, thymus, and in some instances, spleen) were collected from chicks 7-35 days of age. Growth of the chicks fed these diets was monitored over the experimental period as was lymphoid organ growth. The development of the primary lymphoid organs was further assessed by histological techniques and the organ contents of vitamin E (alpha-tocopherol) and Se were determined. Specific deficiencies of either Se or vitamin E were found to significantly impair bursal growth as did a combined deficiency. Thymic growth was impaired only by the combined deficiency diet. Severe histopathological changes in the bursa resulted from the combined deficiency and these were detectable by 10-14 days after hatching. These changes were characterized by a gradual degeneration of the epithelium and an accompanying depletion of lymphocytes. Similar changes, although slower to develop and less severe, were observed in the thymus as a result of the combined deficiency. When both serum and tissue levels of vitamin E and Se were monitored, it was observed that these were rapidly and independently depleted by the specific deficiency diets. These data suggest that the primary lymphoid organs are major targets of Se and vitamin E dietary deficiencies and provide a possible mechanism by which immune function may be impaired.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

Mitogenic action of factors secreted by avian erythroblastosis virus transformed cells

We describe here the capacity of erythroid LSCC HD3 cells, transformed with a ts mutant of avian erythroblastosis virus, to grow in a chemically defined medium without serum at 36 degrees C, but not at 41 degrees C. At this latter temperature the activity of v-erbB oncogene is suppressed. However, cell growth at 41 degrees C could take place either by addition of the medium derived from LSCC HD3 cells grown at 36 degrees C (conditioned medium), or by addition of fetal calf serum. These results show that LSCC HD3 cells, maintained under conditions in which the v-erbB oncogene is active, secrete growth factor(s) which exhibit a mitogenic effect similar to that observed with calf serum.     

Cytotoxicity of cholesterol oxides and their effects on cholesterol metabolism in cultured human aortic smooth muscle cells

Incubation of monolayers of cultured human aortic smooth muscle cells with oxygenated sterols (25-hydroxycholesterol, 7-ketocholesterol, or cholesterol 5,6-epoxide) markedly inhibited growth though the viability of the culture was not affected. The effects on growth was concentration dependent, and 25-hydroxycholesterol was the most potent inhibitor of cellular growth as measured by decreased incorporation of thymidine into DNA and suppression of HMG-CoA reductase activity. The inhibitory effect of 25-hydroxycholesterol on cellular growth was not reversible if the cultures were grown in medium with normal fetal calf serum. However, in medium with delipidated serum, addition of purified cholesterol partially prevented growth inhibition induced by 25-hydroxycholesterol. Purified cholesterol, independently or in combination with tocopherol had no toxic effect on cellular growth. Addition of cholesterol oxides to the incubation medium stimulated lysosomal activation and release of acid phosphatase into the culture medium. The effect was concentration dependent and inversely related to cellular growth.     

The effect of long-term selenium and vitamin E-enriched diet on the content of lipid peroxides and cholesterol in rats

The effect of long-term diets enriched with natural antioxidants was studied on Wistar rats with average initial body weight 150 g. After enrichment of the diet with selenium (0.1 ppm of sodium selenite per 100 g of diet), with vitamin E (6 mg of alpha-tocopherol per 100 g of diet) and selenium and vitamin E together the following results were obtained: diets enriched with selenium or vitamin E given for 12 months reduced the production of lipid peroxides in the liver and serum of the rats. On the other hand, addition of both antioxidants to the diet had no effect on lipid peroxide levels in the animals. Diet enrichment for 12 and 18 months with selenium or vitamin E had no effect on the levels of total cholesterol and HDL cholesterol. The obtained results suggest that selenium and alpha-tocopherol exert an inhibitory action on the processes of ageing in the experimental animal model.     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Effect of lipids on the expression of cell transformation

It has been demonstrated that the Kirsten sarcoma virus-transformed cell line, K3T3, when grown in media containing delipidized fetal calf serum (AES), regained many of the properties of the untransformed parent cell, BALB/3T3. Such properties include morphology, adherence, saturation density, and growth patterns. After growth for several passages in AES, return of K3T3 cells to unextracted fetal calf serum did not restore the transformed phenotype for many generations, although eventually it does return. Cholesterol and linoleic acid added in combination to the growth medium prevented the AES-induced change to the normal, untransformed phenotype. Vitamin E and linoleic acid, on the other hand, did not prevent such a change from taking place.     

Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts.

The effect of various growth factors on the synthesis of hyaluronan in human fibroblasts was investigated. When tested in medium containing 0.5% fetal calf serum, platelet-derived growth factor (PDGF)-BB was found to stimulate hyaluronan synthesis; the maximal response was equal to or higher than that obtained with 10% fetal calf serum. PDGF-AA gave only a limited effect, indicating that the stimulatory effect of PDGF on hyaluronan synthesis was mainly transduced via the B-type PDGF receptor. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 1 also stimulated hyaluronan synthesis; their effects were less than that of PDGF-BB, but combinations of factors produced potent stimulatory effects on hyaluronan synthesis. All factors stimulated hyaluronan synthesis in sparse as well as dense cultures. The effects of the factors on hyaluronan synthesis did not correlate with their mitogenic activities; PDGF-BB, EGF and bFGF are equipotent mitogens, but PDGF-BB had a much more potent effect on hyaluronan synthesis, and TGF-beta actually inhibits the growth of fibroblasts under the conditions of the assay.