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J E Biaglow

Publications and source records attributed to J E Biaglow.

At least 73 records · Page 4Linked to original sources
PubMed1984

On the mechanism for enhancement of tumor radiation to hyperbaric oxygen in sodium pentobarbital anesthetized rodents.

Pentobarbital anesthesia has been observed to increase markedly the effectiveness of respiration of oxygen at 3 atmospheres of pressure absolute to increase the response of early generation isotransplants of C3H mouse tumors to two-dose irradiation. A possible mechanism of this phenomenon is suppression of oxygen utilization by the pentobarbital and hence increasing mean pO2 and oxygen diffusion lengths. Measurements of QO2 of suspension of MCaIV and FSaII cells from freshly excised tumor tissue have been measured for cells suspended in PBS, Hank's buffered with HEPES +/- glutamate. The oxygen utilization by these tumor cells in vitro (when measured at congruent to 10 minutes after excision) is low, viz. 1 nmole/min/mg protein as compared with 6-9 nmoles/min/mg protein for established cell lines cultured in vitro. The suppression of QO2 by 2mM pentobarbital is less than 10%. This is a concentration of pentobarbital that is judged to be close to that which obtains in the tissues of the animals in the radiation response assays. Pentobarbital at .2mM did not change the cell survival characteristics of Chinese V79 cell spheroids irradiated in vitro. The results of these experiments do not indicate the suppression of oxygen utilization is an important contributor to the observed phenomenon of the increased response of tumors irradiated in mice respiring oxygen at high pressure. The role of hypothermia produced by the anesthesia is under further study.

Animals↗
PubMed1984

Factors involved in depletion of glutathione from A549 human lung carcinoma cells: implications for radiotherapy.

We have measured the rate of GSH resynthesis in plateau phase cultures of A549 human lung carcinoma cells subjected to a fresh medium change. Buthionine sulfoximine (BSO) blocks this resynthesis. Diethyl maleate (DEM) causes a decrease in accumulation of GSH. If DEM is added concurrently with BSO there is a rapid decline in GSH that is maximal in the presence of 0.5 mM DEM. GSH depletion rapidly occurs when BSO is added to log phase cultures which initially are higher in GSH content. Twenty-four hr treatment of A549 cells with BSO results in cells that are more radiosensitive in air and show a slight hypoxic radiation response. A 2 hr treatment with either 0.25 mM or 0.5 mM DEM results in some hypoxic sensitization and little increase in the aerobic radiation response. The 24 hr BSO + 2 hr DEM treatment sensitizes hypoxic cells to a greater degree than either agent alone but does not increase the aerobic response more than is seen with BSO alone. Cells treated simultaneously with BSO + DEM show little increase in the hypoxic radiation response, compared to DEM alone, but are more sensitive under aerobic conditions. Decreased cell survival for aerobically irradiated log phase A549 cells occurs within minutes after addition of a mixture of BSO + DEM. The decreased cell survival following aerobic irradiation, after prolonged treatment with BSO or acute exposure to BSO + DEM, may be in part due to inhibition of glutathione peroxidases. For example, glutathione-S-transferase, known to have glutathione peroxidase activity (non-selenium), is nearly completely inhibited by the BSO treatments. In addition, cellular capacity to react with peroxide (glutathione peroxidase, selenium containing) was also inhibited. We suggest that the enhanced aerobic radiation response is related to an inability of GSH depleted cells to inactivate either peroxy radicals or hydroperoxides that may be produced during irradiation of BSO treated cells. Furthermore, enhancement of the aerobic radiation response may be useful in vivo if normal tissue responses are not also increased.

Buthionine Sulfoximine↗
PubMed1984

Depletion of intracellular GSH and NPSH by buthionine sulfoximine and diethyl maleate: factors that influence enhancement of aerobic radiation response.

Many investigators have observed aerobic sensitization of V79, CHO and A549 (human lung carcinoma) cells upon depletion of GSH using buthionine sulfoximine (BSO). Recently we discovered that this aerobic sensitization can be reversed if WR-2721 or N-acetylcysteine is added to the cells just prior to irradiation. Reversal requires that the exogenous thiols be present during the time of irradiation. One possible explanation was that these thiols entered the cells and either increased the pool of cellular nonprotein thiols or reversed the thiol-depleted state by stimulation of GSH synthesis. Cells treated with BSO do not readily regenerate intracellular GSH because this agent irreversibly inhibits gamma-glutamyl synthetase. For A549 monolayer cultures, there is approximately 50% regeneration 6 hr after removal of 0.01 mM BSO, 20% 6 hr after 0.1 mM BSO, and only 5% 6 hr after 0.5 mM BSO. We found that addition of WR-2721 or N-acetylcysteine to BSO-treated cells did not affect the rate of regeneration of intracellular GSH. Thus, reversal of the aerobic sensitization of A549 cells by BSO cannot be explained on the basis of intracellular thiol levels alone, or by rapid reversal of BSO inhibition. In addition, diethylmaleate (DEM)-treated cells are considerably different from BSO-treated cells with respect to the ability to regenerate GSH. After removal of DEM, A549 cells immediately begin GSH resynthesis, and return to control levels occurs within 2 hr. Exogenous 5 mM GSH increases the rate of resynthesis of GSH in DEM-treated cells, but not in BSO-treated cells.

Aerobiosis↗
PubMed1984

Effects of D,L-buthionine-S,R-sulfoximine on cellular thiol levels and the oxygen effect in Chinese hamster V79 cells.

The role of glutathione (GSH) and total non-protein thiols (NPSH) in repairing radiation-induced free radical damage incurred under aerated and hypoxic conditions was investigated using Chinese hamster V79 cells cultured in vitro. GSH and NPSH levels were depleted in V79 cells of varying cell densities using the gamma-glutamyl-cysteine-synthetase inhibitor, D,L-Buthionine-S,R-sulfoximine (BSO). A small change in hypoxic cell radiosensitivity could be attributed to the loss of GSH while depletion of thiols to lower levels affected both aerated and hypoxic cell radiosensitivity, resulting in no change in the OER. Only a long term incubation with BSO produced a large change in the OER, by which time many other biochemical pathways using GSH and amino acids are likely to be affected.

Animals↗
PubMed1984

Chemosensitization: do thiols matter?

It is well known that endogenous sulfhydryls are radioprotective in mammalian cells. Their comparable role in chemotherapeutic drug toxicity has been known for almost as long but less well defined. Thiol depletion as a mechanism responsible for enhanced cytotoxicity of melphalan was assayed by pretreatment of cells in vitro with misonidazole and buthionine sulfoximine (BSO). Hypoxic cell sensitizers, such as MISO, deplete endogenous thiols by metabolic activation under hypoxic conditions to thiol reactive intermediates, whereas BSO specifically inhibits a key enzyme in the synthesis of glutathione. For a given level of thiol reduction, sensitization to melphalan was far greater by preincubation with MISO than it was for BSO. This indicated that thiol reduction itself was not the sole factor involved in chemosensitization by MISO. As evidence that the method of thiol depletion predisposes to the expression of biological damage, it was shown that cells preincubated with MISO were appreciably more vulnerable to oxidative stress than those exposed to BSO. BSO was shown to totally inhibit the repair of damage from a preincubation treatment with MISO, demonstrating that recovery is dependent upon thiol regeneration. Thiol depletion "per se" is a good qualitative but not necessarily a quantitative indicator of chemosensitization--the biological and biochemical function of the thiol depleting agents used influences further drug interactions. The results of the study with these two agents suggest that thiols may play a potentially more critical role in the repair rather than the initiation of drug-induced damage.

Animals↗
PubMed1984

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 McCoy's 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 McCoy's 5A has been compared with that in Ham's 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 McCoy's 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.

Catalase↗
PubMed1984

Factors associated with the preincubation effect of hypoxic cell sensitizers in vitro and their possible implications in chemosensitization.

The enhancement of melphalan toxicity was observed by preincubation of V-79- 379A cells in spinner culture with multiple doses of misonidazole (miso) or SR-2508 under hypoxic conditions. Chemosensitization was shown to be a function of sensitizer concentration and duration of exposure to the alkylating agent. A preincubation exposure of cells with 5 mM miso reduced endogenous cell thiols to less than 5% of controls and enhanced melphalan toxicity by a factor of 4.7. Cells preincubated with miso not only had lower levels of nonprotein thiols, but also were shown to have altered levels of intracellular calcium and a lower threshold to oxidative stress as measured by toxicity to cysteamine or H2O2. Preincubated cells, hypoxic cells, and cells receiving moderate hyperthermia (42.5 degrees C for 3 hr) all showed increased sensitivity to either cysteamine or H2O2. The increased killing of preincubated cells by cysteamine was shown to be similar to that of H2O2, and the dramatic reduction of cysteamine toxicity by catalase indicated H2O2 was the major reaction associated with this effect. These results indicate that preincubated cells exhibit a variety of biological effects that may significantly influence their response to further treatment with drugs or radiation, especially where peroxidative and free radical mechanisms are involved. The depletion of endogenous thiols, calcium disturbance, and vulnerability to oxidative stress are factors to be considered when interpreting mechanisms of combined drug action and effects that may potentially be exploited in terms of therapeutic gains.

Animals↗
PubMed1984

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine.

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.

Animals↗
PubMed1984

Enhancement of cysteamine cytotoxicity by hyperthermia and its modification by catalase and superoxide dismutase in Chinese hamster ovary cells.

Chinese hamster ovary cells were exposed to the sulfhydryl compound cysteamine at concentrations ranging from 0 to 8 mM for 120 min. No toxicity was found in cells maintained at 5 degrees during treatment; however, at 37 degrees and 44 degrees a paradoxical toxicity was observed, i.e., substantial toxicity was observed at cysteamine concentrations of 0.2 to 1 mM but decreased at higher drug concentrations. When drug-treated cells were exposed to a 30-min 44 degrees -heat treatment (surviving fraction, 0.15 in the absence of drug) toxicity was markedly enhanced. At 0.4 mM cysteamine, the surviving fraction was approximately 0.6 at 5 degrees, 0.01 at 37 degrees, and 0.00008 when the 44 degrees -heat treatment was also used. Cysteamine toxicity was not modified by the addition of superoxide dismutase (10 micrograms/ml) but was completely blocked by the addition of catalase (50 micrograms/ml) over the drug concentration range of 0.2 to 2.0 mM. Cysteamine autoxidation as measured by O2 uptake at 0.4 mM proceeds through hydrogen peroxide (H2O2) production as evidenced by the regeneration of O2 upon the addition of catalase. In contrast, at 4.0 mM cysteamine, O2 regeneration was not pronounced. The data suggest that the production of H2O2 is the first reaction step in the mechanism of cysteamine toxicity. The subsequent production of highly reactive oxygen species like hydroxyl radicals (.OH) from H2O2 in the presence of reduced metal (Fenton chemistry) probably leads to the observed cellular toxicity.

Animals↗
PubMed1983

Characterization of the uptake and toxicity of a fluorescent thiol reagent.

3-(4-Maleimidylphenyl)-4-methyl-7-diethylamino coumarin (CPM), is a fluorescent thiol-binding agent. CPM is nontoxic to Chinese hamster V-79 cells (2 x 10(5) cells/ milliliter) exposed to 2.5 micrograms/milliliter for 30 minutes. However, both toxicity and cellular binding were directly dependent on the drug:cell ratio. Using flow cytometry, cellular binding of CPM correlated with inhibition of DNA synthesis. In cells pretreated with the thiol binding drugs N-ethylmaleimide, diamide and diethylmaleate or the carcinogens 4-nitroquinoline-1-oxide and AF-2 (furylfuramide), the subsequent binding of CPM was reduced by 40% or more.

Animals↗
PubMed1983

Cyanide insensitive respiration in mammalian cells: an artifact of mycoplasmal contamination.

Oxygen consumption by mammalian cells occurs primarily in the mitochondria although alternate oxidation pathways are also present. The resistance (often high) of these alternate pathways to metabolic inhibitors like CN- determine the residual oxygen consumption in the presence of the inhibitor. In many plant cells and micro-organisms the cyanide insensitive pathway can account for an appreciable fraction of the total oxygen consumption. We were studying oxidation by electron-affinic drugs in nonmitochondrial pathways and were surprised to find some mammalian cell lines having 10% - 90% residual oxygen consumption in the presence of 1 mM CN-. This oxygen consumption was associated with mycoplasmal contaminants (Mycoplasma hyorhinis, Mycoplasma arginini) and disappeared after treatment with appropriate antibiotics. The rate of CN- insensitive oxygen consumption was dependent on the mammalian cell concentration suggesting that the organisms were oxidizing a product of the mammalian cells. In one species (Mycoplasma hyorhinis) lactate may be the oxidative substrate. Mycoplasmas are known to cause a large number of metabolic alterations in their mammalian cell hosts (Stanbridge et al, 1978) sometimes indirectly due to the metabolism of microorganisms themselves, and sometimes directly due to interference with the normal metabolism of the mammalian cells. It is also known that the respiratory pathways of mycoplasmas are vastly different from those of mammalian cells, (Van Demark and Smith, 1964; see Pollack, 1980 for review) and one might expect alterations of the overall respiratory patterns between infected and non-infected mammalian cell-cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Nonprotein thiols and the radiation response of A549 human lung carcinoma cells.

Glutathione (GSH)-depletion by buthionine sulphoximine (BSO) altered both the aerobic and anaerobic radiation response of A549 human lung cancer cells grown in vitro. The oxygen enhancement ratio (o.e.r) was increased slightly from 3.0-3.3. The lack of an effect of GSH-depletion on o.e.r. reduction, provides a system whereby the mechanism of action of the thiol reactive reagent diethylmaleate (DEM) can be investigated. Pretreatment of cells with DEM, under non-toxic concentrations, removed 13 per cent of the intracellular NPSH and resulted in an o.e.r. of 2. When BSO followed by DEM was used, so that both GSH and NPSH were reduced to zero, an o.e.r. of 1.5 was obtained. Cells treated with 1 mM BSO for 24 hours contained 10 per cent NPSH and no GSH. When these cells were exposed to 0.5 or 1 mM DEM briefly, during irradiation, the o.e.r. was 2.4 and 1.7 respectively. In some cases altered o.e.r.s occurred in combination with increased aerobic responses. This was especially true for aerobic irradiations of BSO-treated cells in the presence or absence of DEM. However, the increased aerobic response was offset by a more dramatic increase in the hypoxic response. These results indicate (a) that GSH plays a significant role in aerobic radiation response but is not a principal factor in o.e.r.-reduction, and (b) that reduction of the o.e.r. by DEM is not due primarily to GSH-removal. The preferential radiosensitization of hypoxic cells by DEM may involve reactions of this compound with NPSH or protein SH, or may be related to the ability of DEM to mimic oxygen as a hypoxic cell radiosensitizer.

Aerobiosis↗