Birgit Jacobson

Factors Influencing the Oxidation of Cysteamine and Other Thiols: Implications for Hyperthermic Sensitization and Radiation Protection

Some of the factors influencing the oxygen uptake and peroxide formation for cysteamine (MEA) and other thiols in serum-supplemented modified McCoys 5A, a well-known medium used to cultivate a variety of cells in vitro, have been studied. The oxidation of MEA and cysteine in modified McCoys 5A has been compared with that in Hams F-12, MEM, and phosphate-buffered saline. All of the growth media were supplemented with 10% calf serum and 5% fetal calf serum. The rate of oxygen uptake for all of the studied thiols was greatest in McCoys 5A. The data indicate that this medium may contain more copper than the other preparations. MEA and cysteine were found to be more effective at 0.4 mM at producing peroxide than dithiothreitol (DTT). N-acetylcysteine was the least reactive. The ability to produce peroxide is dependent upon the temperature, the concentration of thiol, the presence of copper ions, and pH of the medium. MEA and other thiol oxidation is inhibited by the copper chelator diethyldithiocarbamate. Catalase also reduces the oxygen uptake for all thiols. This inhibition involves the recycling of peroxide to oxygen. Superoxide dismutase (SOD) was found to stimulate the oxygen uptake in the case of MEA and cysteine, but had little or no effect with DTT and glutathione. The combined presence of SOD and catalase resulted in less inhibition of oxygen uptake than that obtained by catalase alone. Alkaline pH was found to enhance the oxidation of cysteine and MEA. An important observation was the inhibition of MEA oxidation at 0 degrees C and the stimulation at 42 degrees C. The results indicate that many problems may arise when thiols are added to various media. A major consideration is concerned with the production of peroxide, superoxide, and reduced trace metal intermediates. The presence of these intermediates may result in the production of hydroxyl radical intermediates as well as the eventual oxygen depletion from the medium. Oxygen depletion may alter the results of radiation sterilization and carcinogen activation. Radical production will cause cell damage that is temperature dependent. Therefore, careful consideration must be given to changes in oxygen tension when thiols are added to cells growing in complicated growth medium to protect against either chemical or radiation damage.

THE OXIDATION OF ASCORBATE BY ELECTRON AFFINIC DRUGS AND CARCINOGENS

Abstract— The nitrobenzenes, the carcinogens 4‐nitropyridine‐N‐oxide and 4‐nitro‐quinoline‐N‐oxide as well as the nitrofurans, also known to be carcinogenic, have been found to enhance the reaction of ascorbate with oxygen. The reaction results in the oxidation of ascorbate, the production of dehydroascorbate, superoxide radical, peroxide and water. The drugs are not reduced to stable intermediates during the oxidation but are recycled to their original state. The oxygen consumption is partially inhibited by either superoxide dismutase or catalase. If both superoxide dismutase and catalase are included in the reaction mixture, total oxygen consumption was equal to the amount expected for oxidation of ascorbate to dehydroascorbate and reduction of oxygen to water. The oxygen consumption was inhibited by ferricytochrome c. Semiquinones, nitro and hydroxylamine radicals, produced by electron transfer from ascorbate, reduce ferricytochrome c. These oxygen reactive radicals are responsible for the stimulation of oxygen utilization and ascorbate oxidation. In addition we have found that Ehrlich cells, containing catalase and superoxide dismutase, inhibit the drug catalyzed oxidation of ascorbate. The presence of cyanide, known to inhibit catalase and superoxide dismutase, abolished the cell effect for most of the drugs tested.

[36] Oxaloacetate transcarboxylase from Propionibacterium☆

Publisher Summary This chapter deals with the Oxaloacetate transcarboxylase from propionibacterium. The enzyme activity is assayed spectrophotometrically by determining oxaloacetate formation through coupling with malate dehydrogenase. This assay is used routinely, except when lactate dehydrogenase or DPNH oxidase is present. If the latter are present in small amounts, a control value is determined by omission of methylmalonyl-CoA and is subtracted from the value obtained with the complete assay mixture. When these contaminants are excessive, the reaction is carried out without the addition of malate dehydrogenase and DPNH, and after a suitable interval the mixture is deproteinized with trichloroacetic acid and the oxaloacetate is determined in the neutralized solution using malate dehydrogenase. The direct assay with correction for the control usually can be used for the purification described below and thereafter the control value is so small that it may be neglected. Transcarboxylase is isolated from the bacteria grown in lactate, glucose, or glycerol. The yields of cells and transcarboxylase are quite similar from glucose or glycerol media; glycerol has the advantage that it does not cause caramelization when sterilized with other constituents of the medium, and contamination by other bacteria is less likely with this substrate.

The catalytic effect of the carcinogen “4-nitroquinoline-N-oxide” on the oxidation of vitamin C

Abstract The carcinogen 4-nitroquinoline-N-oxide was found to mediate the reaction between ascorbate and oxygen. The oxidation of ascorbate was initiated by the production of the nitro radical anion which reacted with oxygen to produce the oxygen superoxide radical anion, peroxide and hydroxyl radical. The production of partially reduced oxygen intermediates resulted in additional reactions with ascorbate. The consumption of oxygen could be either completely blocked by reacting the nitro radical with ferricytochrome c or partially blocked by the combined effects of superoxide dismutase and catalase. The consumption of oxygen could be enhanced by reducing the hydroxyl radicals with dimethylsulfoxide.

Factors Influencing the Oxygen Consumption and Radiation Response of Cultured Mammalian Cells

There are published data (Gullino, 1975) that suggest that as a tumor increases in size, its QO2 decreases. Measurements on tumor homogenates in vitro and in vivo perfusion studies indicate large variations in oxidative capacity among various types of tumors (Gullino, 1975). However, we have found that human tumor cells in culture and other cultured mammalian cells have similar oxygen utilization rates (cf. Table I). The differences between tumor cell oxygen utilization in vivo and oxygen uptake for log phase cultures (Table I) suggests that metabolic controls or cellular adaptations are operative in vivo. Similar controls may or may not operate or exist in vitro. Therefore we have investigated a number of factors that might influence cellular oxygen consumption in vitro and of the role that altered oxygen consumption plays in the response of cells to radiation in vitro.

Biocytin as a constituent of methylmalonyl-oxaloacetic transcarboxylase and propionyl CoA carboxylase of bacterial origin☆

Abstract Propionyl-CoA carboxylase containing tritium-labeled biotin has been partially purified from Mycobacterium smegmatis . After preliminary purification the purity of the enzyme can be followed by specific radioactivity. The propionyl-CoA carboxylase of bacterial origin differs from the same enzyme isolated from animal tissues. It has a broader pH range and a greater substrate affinity. Biocytin (ϵ- N -biotinyl- l -lysine) containing tritium-labeled biotin has been isolated and purified from preparations of oxaloacetic transcarboxylase of Propionibacterium shermanii and of propionyl-CoA carboxylase of M. smegmatis . 3

Effect of Calcium Channel Blocking Drugs on Tumor Cell Oxygen Utilization

Oxygen tension in tumor tissue can influence the effectiveness of radiation and many chemotherapeutic agents like misonidazole, bleomycin and adriamycin, which kill tumor cells via oxygen-linked metabolic mechanisms. Therefore it is important to improve tumor oxygenation during the course of treatment with either radiation or drugs. Tumor oxygen tension can be influenced by blood flow as well as by the rate of tumor cell oxygen utilization. We have studied the factors that influence tumor cell oxygen utilization (1,2) in vitro and have previously found that pentobarbital sensitizes tumor cells to hyperbaric oxygen breathing (3). Part of the mechanism may involve either inhibition of oxygen utilization or body temperature lowering, which may also produce reoxygenation by inhibition of tumor cell oxygen utilization (4). We have found that the anaesthetic drugs chlorpromazine and stelazine inhibit cellular oxygen utilization (5). The mechanism of action of these drugs may involve alterations in voltage-dependent calcium channels. Therefore we investigated further the possibility that calcium channels were involved in cellular respiration. We have studied the currently used heart attack drugs diltiazem, nifedipine (procardia) and verapamil as well as praziquantel for their effects on tumor cell oxygen utilization in vitro. We have also determined their inhibiting capacity with tumor cells incubated in the presence of succinate and glucose, substrates known to stimulate, in the former case, and to inhibit cellular oxygen utilization in the later case.

Control of Oxygen Utilization In Vitro and In Vivo: Implications for Radiotherapy of Tumors

Several studies have shown that as a tumor increases in size, its QO2 decreases1,2. Measurements on tumor homogenates in vitro and in vivo perfusion studies, indicate large variations in oxidative capacity among various types of tumors3. However, we have previously found that human tumor cells in log-phase culture as well as log-phase cultures of mammalian cells have similar oxygen consumption rates4. Some cultured lines show a decrease in oxidative capacity in plateau phase growth5,6. The differences between QO2 values obtained for in vitro versus in vivo cells as well as the differences between log and plateau phase growth suggest that metabolic controls or cellular adaptations are operative in vivo. Similar controls may or may not operate or exist in vivo. Tberefore we have initiated a study of the possible factors that might influence cellular oxygen consumption for a number of murine tumors including the mouse mammary carcinoma (MCaIV), a fibrosarcoma (FSaII), a squamous cell carcinoma (SCCaVII), the Ehrlich ascites tumor and the rat brain carcinoma (9L). We have compared the tumor rates with cultured FSall as well as HEp-2, the A549, a human lung carcinoma and the V79 Chinese hamster lung cell.

Cellular Oxygen Utilization and Hypoxia: Interaction of Dithiols with Cellular Electron Transfer Systems

DL-Dithiothreitol (DTT) better known as Cleland’s reagent, and dithioerythritol (DTE) are isomers of 2,3-dihydroxy-l-4-dithiobutane which were originally used by Cleland (1964) as antioxidants (1). They have found wide spread use in chemistry, biochemistry, biology and medicine, principally as reductants for disulfides as well as protectors of sulfhydryls against oxidants. DTT has found application as a chemical that protects microbes (2) and mammalian cells (3,4) against the damaging effects of ionizing radiation. It is this latter property that has been of interest to us. The radioprotection conferred upon cells by DTT may be due to its stimulation of cellular oxygen utilization. Increases in oxygen consumption may produce hypoxic cells in multicellular systems (5) as well as create intracellular hypoxia (4,5). The lowering of oxygen concentration results in protection against radiation damage due to the well known oxygen effect (6). In addition to effects on oxygen utilization, dithiols also react spontaneously with oxygen to produce superoxide and peroxide. This reaction is enhanced in the presence of copper and iron that are normally found in the growth medium of cultured cells. Depletion of oxygen by this spontaneous reaction will also cause hypoxia and radiation protection (7).