Katsuhiro Kageyama

Enhanced Protection Against Peroxidation-Induced Mortality of Aortic Endothelial Cells by Ascorbic Acid-2-O-Phosphate Abundantly Accumulated in the Cell as the Dephosphorylated Form

Bovine aortic endothelial BAE-2 cells exposed to the peroxidizing agent, tert-butylhydroperoxide (t-BuOOH) or 2,4-nonadienal (NDE), suffered from disruption of cell membrane integrity and from reduction of mitochondrial dehydrogenase activity as assessed by fluorometry using ethidium homodimer and photometry using WST-1, respectively. The cells were protected from t-BuOOH-induced injury more markedly by L-ascorbic acid-2-O-phosphate (Asc2P) stably masked at the 2,3-enediol moiety, which is responsible for the antioxidant ability of L-ascorbic acid (Asc), than by Asc itself. In contrast, NDE-induced membrane disruption but not mitochondrial dysfunction was prevented by Asc2P, whereas Asc exhibited no prevention against both types of injury. The amount of intracellular Asc was 7.2- to 9.0-fold larger in Asc2P-administered BAE-2 cells, where the intact from Asc2P was not detected, than in Asc-administered cells as assessed by HPLC of cell extract with detection by coulometric ECD and UV. During transmembrane influx into the cell, Asc2P was concentrated as highly as 70- to 90-fold relative to the extracellular Asc2P concentration, whereas Asc was 8- to 13-fold concentrated as estimated based on an intracellular water content of 0.59 pL/cell determined by [14C]PEG/gas chromatography. Thus, Asc2P but not Asc is highly concentrated in the aqueous phase of the cell after prompt dephosphorylation, and may thereby render the cell more resistant to t-BuOOH-peroxidation assumedly via scavenging of intracellular reactive oxygen species than to peroxidation with the less hydrophilic agent NDE.

Altered production of the active oxygen species is involved in enhanced cytotoxic action of acylated derivatives of ascorbate to tumor cells

Our previous study shows that 6-O-acyl derivatives of L-ascorbic acid inhibits more markedly cell growth of mouse Ehrlich carcinoma than ascorbic acid. The present study shows that 6-O-palmitoyl ascorbic acid but not ascorbic acid prolongs the lifespan of mice into which tumors such as Meth A fibrosarcoma, MM46 mammary carcinoma, Ehrlich carcinoma and sarcoma 180 are implanted. The potentiated cytotoxicity of 6-O-palmitoyl ascorbic acid is not due to an increase in duration time of the cytotoxic action, because 6-O-palmitoyl ascorbic acid is gradually inactivated during contact with tumor cells and exhibits a similar action time curve to that of ascorbic acid as shown by clonal growth assay. Cytotoxicity of 6-O-palmitoyl ascorbic acid is markedly diminished by combined addition of catalase and superoxide dismutase (SOD), as shown by dye exclusion assay, whereas the cytotoxicity was slightly reduced by either enzyme alone but not by the specifically inactivated or heat-denatured enzymes. In contrast, cytotoxicity of ascorbic acid is abolished by catalyse but not SOD. Autooxidation of 6-O-palmitoyl ascorbic acid was not inhibited by catalase plus SOD. The results indicate that cytotoxicity of 6-O-palmitoyl ascorbic acid is attributed at least partly to both hydrogen peroxide (H2O2) and superoxide (O2-.) generated at the early stage. Cytotoxicity of 6-O-palmitoyl ascorbic acid is also appreciably attenuated by singlet oxygen (1O2) scavengers such as hydroquinone, 1,4-diazobicyclo-2,2,2-octane or sodium azide, but not by hydroxyl radical scavengers including butylated hydroxytoluene, D-mannitol, benzoic acid and ethanol. Thus, in contrast to cytotoxicity of ascorbic acid mediated entirely by H2O2 initially generated, acylated ascorbic acid produces a diversity of active oxygen species including H2O2, O2-. and other species secondarily generated via disproportion, which may be additively involved in the enhanced cytotoxic action.

The maximum and minimum water content and cell volume of human erythrocytes in vitro

The maximum and minimum water contents of human erythrocytes were measured after exposure to various osmotic pressures. Within a range of osmolarities, at which no haemolysis occurred, the water content reached its maximum, 78.1%, at 180 mosM and its minimum, 54.8%, at 800 mosM. Simultaneously, the mean cell volume increased to 98.5 microns 3 at 180 mosM and decreased to 77.2 microns 3 at 800 mosM.

Moderately controlled transport of ascorbate into aortic endothelial cells against slowdown of the cell cycle, decreasing of the concentration or increasing of coexistent glucose as compared with dehydroascorbate

Uptake of L-[1-14C]ascorbic acid (Asc) of 12.5-200 µM for 1 h intobovine aortic endothelial BAE-2 cells grown to confluence was as low as43-64% (per cell) of uptake into the cells grown to nearly one-fourthconfluence. [14C]Asc undergoing transmembrane uptake was concentrated andaccumulated in the cell less efficiently ([Asc]in/ex = 8-13) at confluencethan at subconfluence ([Asc]in/ex = 15-24). The declined Asc uptake atconfluence is attributable to slowdown of the cell cycle, because a similardecrease in [Asc]in/ex was shown by subconfluent cells precultured inserum-insufficient medium, resulting in an increase in G1 phase andconcurrent decreases in S and G2 + M phase distributions as determined byflow cytometry. [1-14C]Dehydroascorbic acid (DehAsc) was taken up andaccumulated as Asc, after metabolic reduction, without detectable DehAsc.The [Asc]in/ex values for DehAsc at confluence were as low as 15-69%of those at subconfluence in contrast to the values as retentive as62-75% for Asc, suggesting the moderate control of Asc uptake againstslowdown of the cell cycle. At either confluence or subconfluence,dose-dependence for DehAsc uptake was more marked than for Asc uptake asshown by an uphill slope in a curve of doses versus [Asc]in/ex for DehAsc incontrast to a downhill slope for Asc, suggesting the moderate control forAsc uptake against fluctuation of the dose. Increasing of coexistent glucoseof 5 mM to 20-40 mM, plasma concentrations in diabetic patients, declinedDehAsc uptake to 46-48%, which was less moderately controlled thanAsc uptake retained to 59-73%. Asc uptake did not compete with DehAscuptake, suggesting different transporter proteins for Asc and DehAsc. Thus,Asc uptake into the aortic endothelial cells is more moderately controlledagainst slowdown of the cell cycle, decreasing of the extracellularconcentrations or increasing of coexistent glucose than DehAsc uptake,suggesting a homeostatic advantage of Asc over DehAsc in terms of retentionof intracellular Asc contents within a definite range.

Enhanced inhibition of DNA synthesis and release of membrane phospholipids in tumour cells treated with a combination of acylated ascorbate and hyperthermia

Combined antitumour effects of mono- or diacyl ascorbates and heat treatment were studied in comparison with the parent compound, L-ascorbic acid (AsA). At 37 degrees C, 75 microM 6-O-palmitoyl (6P) and 6-O-stearoyl (6S) ascorbates appreciably inhibited DNA synthesis in Ehrlich ascites tumour cells. Hyperthermia at 42 degrees C for 1 h increased the inhibition. In contrast, AsA or 2,6-O-dipalmitoyl ascorbate (DP), even at concentrations as high as 100 microM, caused no inhibition at 37 degrees C or 42 degrees C. The results suggest that the inhibitory action is not caused by the fatty acid moiety itself; it is more likely to be caused by the balance in the hydrophobicity and hydrophilicity of the monoacylated AsA, a property not found in diacylated or intact AsA. Inhibition of DNA synthesis caused by exposure to 6P during hyperthermia of tumour cells was greater than before or after hyperthermia. 6P or 6S, but not AsA or DP, released phospholipids such as phosphatidylcholine and phosphatidylethanolamine from cells labelled with [14C]oleic acid, as shown by radiocurves taken from thin-layer chromatograms. Damage of the cell membrane seemed to be involved in the inhibition of DNA synthesis caused by monoacylated AsA, which is surface-active.

Studies on in vitro effect of free fatty acids on water content and osmotic fragility of rabbit (lepus cuniculus) erythrocytes

The effects of free fatty acids in the blood on osmotic fragility and water content of erythrocytes when combined with hyperthermia were investigated. The isotonic buffer, containing the fatty acid, was added to the erythrocyte suspension to a final concentration of 200 microM/L. The samples were kept for one hour in an incubator at 37 degrees C or 42 degrees C. The osmotic fragility of erythrocytes was determined by coil planet centrifuge system and intracellular water content was measured by gas-liquid chromatography. A high concentration of unsaturated fatty acid and hyperthermia caused the increase in intracellular water and the decrease in osmotic resistance of the red blood cells compared to saturated fatty acid and the control. The present experiment demonstrated that unsaturated fatty acid was one of the principal chemical and metabolic factors which caused sports anemia.

Control by hyperthermia of ornithine decar☐ylase in Ehrlich ascites tumor cells

The effect of hyperthermia on the activity and the messenger RNA levels of ornithine decarboxylase (ODC), which has a rapid rate of turnover in cultured cells, was studied in Ehrlich ascites tumor cells. When the cells were incubated at 42 degrees C, elevation of ODC activity by a change of the medium was prevented. Total RNA was isolated from cells treated at 37 degrees C or 42 degrees C, and the relative abundance of the ODC mRNA was measured by Northern blot analysis. These levels in heat-treated cells were comparable to those in control cells. Inhibition by hyperthermia was reversible. The recovery was suppressed by cycloheximide but not by actinomycin D. In hyperthermic-treated cells, the biological half-life of ODC was 14 min, which was the same time as for cells cultured at 37 degrees C. These results suggest that hyperthermic treatment of Ehrlich ascites tumor cells suppressed ODC induction during translation, not during transcription or after translation.

Promotive action of acylated ascorbate on cellular DNA synthesis and growth at low doses in contrast to inhibitory action at high doses or upon combination with hyperthermia

Effects of 6-O-palmitoyl ascorbate (ascorbate) developed to increase the antitumour activity of ascorbic acid on DNA synthesis and proliferation of Ehrlich ascites tumour cells were investigated. Treatment of the cells with the acylated ascorbate at 25–50 μM for 1 h resulted in no effect on DNA synthesis, assayed by pulse incorporation of [3H]thymidine after a culture period of 20 h, but led to 49%–87% enhanced DNA synthesis after 4 days, suggesting that long-term culture is required for promotion by ascorbate to occur. At a dose as high as 75 μM acylated ascorbate, however, cellular DNA synthesis was 64% inhibited after 20 h and 99% after 4 days. The results suggest that acylated ascorbate exhibits a dual action on DNA synthesis: promotion at low doses and inhibition at high doses, both of which are potentiated in a time-dependent manner. In contrast to the above-mentioned results at 37°C, acylated ascorbate at 25–75 μM inhibited but did not promote DNA synthesis at 42°C whatever the culture period. Similar results were exhibited when proliferation of cells cultured for a long period was investigated. At 37°C, 50μM acylated ascorbate increased the number of the cells to 3.6 times the control values after 8 days and to 1.9 times after 11 days; in contrast, a 75-μM dose decreased the cell number considerably. Combination with hyperthermia (42°C) suppressed the increase and cell growth was completely inhibited at 75μM.

Effect of hyperthermia on spermidine/spermine N1-acetyltransferase activity in Ehrlich ascites cells

When Ehrlich ascites tumor cells were incubated at 42 degrees C, the activity of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, decreased to 20% of the control level within 1 hr. Protein synthesis, judged from the incorporation of [3H]leucine into the acid-insoluble fraction, was less affected by heat exposure. The decrease in the enzyme activity caused by heat treatment could be reversed by switching the incubation temperature back to 37 degrees C. The reversion of enzyme activity was suppressed by cycloheximide but not by actinomycin D.

A new, rapid and precise method for measuring the intra-cellular water content by gas chromatography and 3H-sucrose.

A new rapid method for measuring intra-cellular water content by gas chromatography and an isotopic (3H-sucrose) technique is described. Water content of circulating red cells was revealed to be 71.26±0.31%. Intra-cellular water content increased to 102.9% of its original value following addition of 3 volumes of acid-citrate-dextrose (ACD) to 20 volumes of blood.