Biomedical subjects

J E Biaglow

Publications and source records attributed to J E Biaglow.

At least 37 records · Page 2Linked to original sources

PubMed1996

Role of hydroxyl radicals in radiation-induced activation of lyn tyrosine kinase in human B-cell precursors.

Here we show that exposure of human B-cell precursors to gamma-rays stimulates the enzymatic activity of the Src protooncogene family protein tyrosine kinase LYN. LYN activation in irradiated cells is not triggered by DNA damage or a nuclear signal since gamma-rays effectively stimulated LYN kinase in enucleated B-cell precursors as well. LYN activation in irradiated cells was abrogated by presence of the OH* radical scavenger dimethylsulfoxide and exposure of intact or enucleated B-cell precursors to chemically generated OH* radicals instead of gamma-rays also triggered LYN kinase activation and enhanced tyrosine phosphorylation of multiple electrophoretically distinct protein substrates. Thus, OH* radicals appear to be both mandatory and sufficient for radiation-induced LYN kinase activation in irradiated B-cell precursors. We further present evidence which indicates that OH* radicals activate LYN by a novel mechanism which involves disruption of inactive LYN-LYN homodimers and monomerization of the LYN kinase after proteolytic degradation of a putative LYN-associated adapter protein through a cytoplasmic TPCK-sensitive chymotrypsin-like protease following its oxidation. LYN kinase plays a pivotal role in initiation of signal cascades that affect the proliferation, differentiation, and survival of B-cell precursors. Our results prompt the hypothesis that a growth regulatory balance might be altered in human B-cell precursors by radiation-induced stimulation of LYN kinase.

B-Lymphocytes↗

PubMed1996

Role of Fenton chemistry in thiol-induced toxicity and apoptosis.

Under certain conditions, many radioprotective thiols can be toxic, causing loss of colony-forming ability in cultured mammalian cells in a biphasic fashion whereby the thiols are not toxic at high or low concentrations of the drug, but cause decreased clonogenicity at intermediate (0.2-1.0 mM) drug levels. This symposium paper summarizes our studies using dithiothreitol (DTT) as a model thiol to demonstrate the role of Fenton chemistry in thiol toxicity. The toxicity of DTT in V79 cells has several characteristics: it is dependent on the medium used during exposure of cells to the drug; the toxicity is decreased or prevented by addition of catalase exogenously, but superoxide dismutase has no effect; the toxicity is increased by addition of copper, either free or derived from ceruloplasmin in serum; and the toxicity can be modified intracellularly by altering glucose availability or pentose cycle activity. Thus the data are consistent with a mechanism whereby DTT oxidation produces H2O2 in a reaction catalyzed by metals, predominantly copper, followed by reaction of H2O2 in a metal-catalyzed Fenton reaction to produce the ultimate toxic species, .OH. Studies comparing 12 thiols have shown that the magnitude of cell killing and pattern of dependence on thiol concentration vary among the different agents, with the toxicity depending on the interplay between the rates of two reactions: thiol oxidation and the reaction between the thiol and the H2O2 produced during the thiol oxidation. The addition of other metals, e.g. Zn2+, and metal chelators, e.g. EDTA, can also alter DTT toxicity by altering the rates of thiol oxidation or the Fenton reaction. Recent studies have shown that in certain cell lines thiols can also cause apoptosis in a biphasic pattern, with little apoptosis at low or high drug concentrations but greatly increased apoptosis levels at intermediate (approximately 3 mM) thiol concentrations. There appears to be a good correlation between those thiols that cause loss of clonogenicity and those that induce apoptosis, suggesting similar mechanisms may be involved in both end points. However, thiol-induced apoptosis is not prevented by addition of exogenous catalase. These observations are discussed in relation to the possible role of Fenton chemistry in induction of apoptosis by thiols.

Animals↗

PubMed1996

Role of guanosine triphosphate in ferric ion-linked Fenton chemistry.

We measured the production of reactive hydroxyl radical (OH.) by Fe2+ itself or complexed with nucleotide triphosphates or tripolyphosphate (TPP). Coumarin-3-carboxylic acid (3-CCA) reacts with the OH. produced by Fe2+, Fe3+ or Cu2+ plus ascorbate and with various iron complexes. We measured in real time the increased fluorescence of 3-CCA after hydroxylation to 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). Phosphate-buffered solutions do not affect the yield of Fe(2+)-linked OH. as do other organic buffer solutions. Our results show that guanosine triphosphate enhances the Fe(2+)-linked production of OH.. We also tested inosine triphosphate, adenosine triphosphate and xanthine triphosphate for their capacity to produce OH. with Fe2+. Inosine triphosphate is the most effective nucleotide in the production of OH.. However, the Fe(2+)-mediated yield of OH. is greater in the presence of TPP compared to the nucleotide triphosphates. Organic buffers as well as the purine and ribose portion of nucleotides compete for OH. and decrease the yield of fluorescent 7-OHCCA. We also decreased the yield of OH. by adding guanosine to the Fe2+/TPP-generating system. Adenosine, ribose and deoxyribose also react with Fe(2+)-generated OH.. The decreased yield of 7-OHCCA occurs because the ribose and purine part of the molecule reacts with OH.. The maximal production of reactive OH., compared to all nucleotides and phosphates tested, occurs with a ratio of 2 TPP/Fe2+ complex. In conclusion, the real-time measurement of the production of fluorescent 7-OHCCA provides a convenient means for measuring chemically generated OH.. The TPP/Fe(2+)-generating mixture, in the presence of 3-CCA, can be used to study the scavenging ability of other competing molecules.

Animals↗

PubMed1996

The importance of choice of anaesthetics in studying radiation effects in the 9L rat glioma.

In the present study we demonstrate that the glycolysis of the tumour 9L glioma, in vivo, may be manipulated with ketamine/xylazine combinations of anaesthetics. Xylazine alone or in combination with ketamine causes hyperglycaemia which is enhanced by glucose injections. Intracellular tumour pH is acidified when glucose is administered with ketamine/xylazine. However, the combination of inorganic phosphate and insulin with ketamine/xylazine and glucose caused an alkaline shift in the tumour pH as measured by 31P NMR. The anaesthetic combination of ketamine/acepromazine did not produce alterations in blood glucose or in tumour pH status as detected by 31P NMR spectroscopy. These results demonstrate dramatic effects of ketamine/xylazine on the acidification or alkalinisation of the cells of 9L glioma. These altered metabolic states are of potential therapeutic importance. The choice of xylazine alone would be useful for chemotherapy and hyperthermia modalities, both known to be dependent upon glucose metabolism and resultant acidification.

Anesthetics↗

PubMed1994

Protection against SR 4233 (Tirapazamine) aerobic cytotoxicity by the metal chelators desferrioxamine and tiron.

PURPOSE: Metal chelating agents and antioxidants were evaluated as potential protectors against aerobic SR 4233 cytotoxicity in Chinese hamster V79 cells. The differential protection of aerobic and hypoxic cells by two metal chelators, desferrioxamine and Tiron, is discussed in the context of their potential use in the on-going clinical trials with SR 4233. METHODS AND MATERIALS: Cytotoxicity was evaluated using clonogenic assay. SR 4233 exposure was done in glass flasks as a function of time either alone or in the presence of the following agents: superoxide dismutase, catalase, 5,5-dimethyl-1-pyrroline, Trolox, ICRF-187, desferrioxamine, Tiron (1,2-dihydroxybenzene-3,5-disulfonate), and ascorbic acid. Experiments done under hypoxic conditions were carried out in specially designed glass flasks that were gassed with humidified nitrogen/carbon dioxide mixture and with a side-arm reservoir from which SR 4233 was added to cell media after hypoxia was obtained. Electron paramagnetic resonance studies were also performed. RESULTS: Electron paramagnetic resonance and spectrophotometry experiments suggest that under aerobic conditions SR 4233 undergoes futile redox cycling to produce superoxide. Treatment of cells during aerobic exposure to SR 4233 with the enzymes superoxide dismutase and catalase, the spin trapping agent DMPO, the water-soluble vitamin E analog Trolox, and the metal chelator ICRF-187 provided little or no protection against aerobic SR 4233 cytotoxicity. However, two other metal chelators, desferrioxamine and Tiron, afforded significant protection against aerobic SR 4233 cytotoxicity (protection factors at 50% survival were 3.8 and 3.1, respectively), while exhibiting minimal protection to hypoxic cells treated with SR 4233. CONCLUSIONS: One potential mechanism of aerobic cytotoxicity is redox cycling of SR 4233 with molecular oxygen resulting in several potentially toxic oxidative species that overburden the intrinsic intracellular detoxification systems such as superoxide dismutase, catalase, and glutathione peroxidase. This study identifies two metal chelating agents, desferrioxamine and Tiron, that were able to protect against aerobic but not hypoxic SR 4233 cytotoxicity.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed1994

Bioreductive metabolism of SR-4233 (WIN 59075) by whole cell suspensions under aerobic and hypoxic conditions: role of the pentose cycle and implications for the mechanism of cytotoxicity observed in air.

PURPOSE: Measurement of pentose cycle (PC) activity is shown to be a noninvasive means for monitoring the reduction of SR-4233 in whole cells. Comparing these measurements to the actual measurements of drug loss under aerobic and hypoxic conditions helps to define the mechanism for the associated aerobic toxicity. METHODS AND MATERIALS: SR-4233 is activated to a toxic species by bioreductive metabolism. NADPH is required for the activation of the drug by purified enzymes, cell homogenates and whole cells. In vivo the NADPH:NADP+ ratio is maintained by the oxidation of glucose via the oxidative limb of the pentose cycle. By measuring radiolabeled 14CO2 released as a product of this oxidation one can get an accurate measurement of the rate of drug metabolism in whole cells. These results are compared to measurements of drug consumption under aerobic and hypoxic conditions using an HPLC assay. RESULTS: SR-4233 stimulates pentose cycle activity to a greater extent in air then under hypoxia, however, in the presence of added catalase, pentose cycle activity is stimulated to a similar extent under both conditions. The higher levels of PC activity observed in air are due to the production of hydrogen peroxide by the nitroxide free radical undergoing futile redox cycling. The contribution of H2O2 to the observed aerobic cytotoxicity of SR-4233 is minimal however, since toxicity is only slightly reduced in the presence of exogenous catalase and antioxidants such as vitamin E. The level of PC stimulation by SR-4233 suggests that the rate of electron addition to the drug is independent of O2 concentration. The loss of drug from the incubation medium, i.e., conversion to a stable intermediate species, occurs approximately five times faster under nitrogen than in air for A549 cells. It is the rate of drug loss from the cell and not the rate of reduction which best correlates with the observed aerobic and hypoxic toxicity. CONCLUSION: Toxicity in air and in nitrogen is directly related to the rate of drug reduction, i.e., at equivalent levels of drug loss we observe equal levels of cytotoxicity.

Aerobiosis↗

PubMed1994

Mechanisms for the oxygen radical-mediated toxicity of various thiol-containing compounds in cultured mammalian cells.

When Chinese hamster V79 cells are exposed to various thiol compounds in phosphate-buffered saline (PBS), some compounds cause toxicity (loss of colony formation), although the dependence on drug concentration and the magnitude of the cell killing vary between the different thiols. For example: dithiothreitol (DTT) and WR-1065 cause a biphasic toxicity whereby cell killing occurs at about 0.2 to 1.0 mM thiol, but is not seen at higher or lower drug concentrations; N-acetylcysteine (NAC) is toxic only at concentrations > or = 2 mM and shows no biphasic pattern; and glutathione (GSH) and penicillamine are only minimally toxic at all concentrations. The effect of the addition of 1 microM Cu2+ to the thiol also depends on the particular thiol: e.g., Cu2+ increases cell killing in the biphasic pattern with WR-1065; it increases the toxicity of NAC only at high thiol concentrations; and it elicits a slight toxicity in the biphasic pattern by GSH and penicillamine. In all cases tested, if the thiol is toxic, the cell killing can be decreased or prevented by addition of catalase, consistent with the hypothesis that the toxicity is mediated through H2O2 produced during the thiol oxidation. However, when the oxidation rates of the various thiols in PBS without and with Cu2+ were measured, the data did not show a simple correlation between the toxicity of the various thiols and their oxidation rates. The rate of the reaction of the various thiols with H2O2 was also determined and showed a better, but still not good, correlation with toxicity. However, cell killing by the various thiols correlated better with the ratio between the half-lives for thiol oxidation and reaction of thiol with H2O2 than with either reaction rate alone. This suggests that the toxicity pattern and magnitude of cell killing in V79 cells by various thiols depend on the interplay between the rate of thiol oxidation and the rate of reaction between the thiol and the H2O2 produced in the thiol oxidation.

Acetylcysteine↗

PubMed1993

Role of copper in the oxygen radical-mediated toxicity of the thiol-containing radioprotector dithiothreitol in mammalian cells.

The thiol radioprotector dithiothreitol (DTT) causes biphasic toxicity in V79 cells: exposure to DTT causes loss of clonogenic cell survival at intermediate concentrations of the drug, but is not toxic at lower (< 0.05 mM) or higher (> 2.0 mM) concentrations. Toxicity depends on the medium in which cells are exposed to DTT. Cell killing is less when cells are exposed to DTT in phosphate-buffered saline (PBS) than when in complete, serum-containing medium, and there is no cell killing at any drug concentration when cells are in serum-free medium. When cells are exposed to DTT in PBS containing 10% fetal calf serum (FCS) or 10% dialyzed FCS, cell killing is increased compared to the response in PBS alone, suggesting that some component(s) of serum is involved in DTT toxicity. Addition of micromolar quantities of copper as either free Cu2+ or ceruloplasmin to serum-free medium increases the toxicity of DTT, but addition of free iron or transferrin has no effect. H2O2 is produced during DTT oxidation and appears to be involved in the toxicity of DTT, because toxicity can be prevented by catalase. H2O2 is also toxic to V79 cells in a medium-dependent fashion, but the toxicity is not influenced by addition of copper, ceruloplasmin, iron or transferrin. The rate of DTT oxidation is also medium-dependent and is increased by copper or ceruloplasmin, but not by iron or transferrin. The data are consistent with the hypothesis that the toxicity of DTT is caused by the copper-catalyzed oxidation of DTT, forming H2O2 which, in turn, produces .OH, the ultimate toxic agent, via the Fenton reaction.

Animals↗

PubMed1993

Role of the pentose cycle in oxygen radical-mediated toxicity of the thiol-containing radioprotector dithiothreitol in mammalian cells.

We have shown previously that the thiol-containing radioprotector dithiothreitol (DTT) kills V79 cells in a manner that is dependent on both the concentration of DTT and the medium. The results are consistent with the hypothesis that DTT toxicity is caused by the copper-catalyzed oxidation of DTT, forming H2O2, which in turn produces .OH, the ultimate toxic species, via the metal-catalyzed Fenton reaction. Because it is known that the pentose cycle plays a role in the ability of cells to deal with oxidative stress, the hypothesis that the pentose cycle is involved in the response of cells to DTT is tested in this paper. The results show that toxicity of both DTT and H2O2 in V79 cells is greater in cells exposed to the drugs in medium lacking glucose than in cells in medium containing glucose. Addition of glucose to medium or buffer lacking it decreases DTT- and H2O2-induced cell killing. Studies using cells deficient in glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose cycle, show that cells of the mutant cell lines (E16 and E48) are more sensitive to cell killing by both DTT and H2O2 than are the parental CHO K1D cells when exposed to the drugs in medium containing glucose. However, toxicity does not differ significantly among the three cell lines when they are exposed to DTT or H2O2 in phosphate-buffered saline that lacks glucose. Measurements of pentose cycle activity show that the pentose cycle in K1D cells is stimulated by DTT, with the pattern and drug concentration dependence of the stimulation being similar to that for cell killing. However, the pentose cycle is not stimulated by DTT in G6PD-deficient cell lines. The data are consistent with the hypothesis that the pentose cycle is one of the cellular pathways that mediates the oxidative stress imposed by DTT or H2O2.

Animals↗

PubMed1992

The importance of peroxide and superoxide in the X-ray response.

Radiation produces a number of damaging radicals as well as peroxide. The chief cellular protection against these radicals, their secondary reactants and peroxide is the cellular glutathione (GSH), GSH peroxidase, GSH-S-transferase (GSHTase), and catalase enzymes. Inhibition of cellular catalase alone does not enhance the aerobic radiation response because cellular GSH peroxidase is equally effective in reducing peroxide. However, inhibition of GSHTase, and partial inhibition of peroxidase by L-buthionine sulfoximine (LBSO)-linked GSH depletion, results in an increased aerobic radiation response. The major pathway for peroxide reduction is the GSH peroxidase. The enzyme is accountable for 70% inactivation of low peroxide concentrations. Catalase accounts for the remaining inactivation. However, it is difficult to assess the relative contributions of GSHTase and peroxidase to the inactivation of radiation-produced hydroperoxides. Our data suggest that GSH depletion results in the inhibition of cellular GSHTase before it inhibits GSH peroxidase. Therefore, part of the increased aerobic radiation response maybe due to cellular inability to reduce hydroperoxides. Peroxide is not a substrate for GSHTase. However, total inhibition of peroxidase by L-BSO plus N-ethylmaleimide (NEM) treatment maximizes the aerobic radiation response. Total inhibition of GSH-S-transferase and peroxidase would block both peroxide and hydroperoxide reduction.

Catalase↗

PubMed1992

Sensitivity to chemical oxidants and radiation in CHO cell lines deficient in oxidative pentose cycle activity.

In this paper we examine the susceptibility of a series of G6PD- CHO cell lines to a variety of chemical oxidants. Addition of these drugs to K1D, the parental cell line, results in as much as a 20-fold increase in pentose cycle (PC) activity over control values. In two of our mutant lines, E16 and E48, little or no stimulation of PC activity is seen. These lines are shown to be much more susceptible to the toxic effects of the chemical oxidants t-butyl hydroperoxide and diamide. PC activity is also stimulated by ionizing radiation in K1D cells. One of the G6PD- cell lines has an increased aerobic radiation response compared to the parental line. However, since this is not the case with the other G6PD- cell lines, it is unclear whether this represents a difference in the absolute value of PC activity or some additional variable that may be influencing the results.

Animals↗

PubMed1991

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate.

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.

Animals↗

PubMed1989

Effect of varying the concentration of damage restituting species in the radical repair model.

From analytical expressions derived for the radical-repair (competition) model describing the relationship between cellular radiosensitivity and oxygen concentration, "K-curve" behavior has been quantified as a function of the concentration of the species S which restitutes the radiation-induced radicals to their original molecular configuration. If these species are identified with thiols, K-curves modified by fractionally depleting [S] through calculation can be compared with experimental data where cells have their thiols depleted using various means, for example, by chemical agents or by the use of cells with decreased thiols because of genetic deficiency. Families of curves have been calculated related both to the S-depleted and the non-S-depleted hypoxic control, the latter of which is used to calculate enhancement ratios. Comparison of the model with experimental data is made.

Animals↗

PubMed1989

Role of glutathione in the aerobic radiation response.

We will review the relationships between glutathione (GSH), protein thiols, and cellular responses to radiation, peroxides, and peroxide-producing drugs. Our primary interest involves the behavior of sulfhydryls as electron and hydrogen carriers, and their capacity to protect various target molecules against radiation and peroxidative damage. We used reagents such as L-buthionine sulfoximine (LBSO), alone and in combination with N-ethyl maleimide (NEM), diamide, and dimethylfumarate, to decrease GSH so that it could no longer participate in the electron transfer reactions. Our results indicate that aerobic sensitization produced by GSH depletion can be further enhanced if electron-accepting agents, such as tertiary butyl hydroperoxide (t-BOOH), are present during irradiation. Hydroperoxide is a substrate for glutathione peroxidase and diverts electrons and hydrogen away from target molecules during its reduction. Sensitivity to radiation seems to be due to the inhibition of the mitochondria's capacity to reduce hydroperoxide. We will also report the mitochondria's ability to reduce the oxygen radicals produced by radiation and drugs. Data also indicate that t-BOOH oxidizes protein thiols which are enzymatically involved in repair of DNA damage.

Aerobiosis↗

PubMed1989