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M E Varnes

Publications and source records attributed to M E Varnes.

54 records · Page 3Linked to original sources
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

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↗
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

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↗
PubMed1983

The role of thiols in cellular response to radiation and drugs.

Cellular nonprotein thiols (NPSH) consist of glutathione (GSH) and other low molecular weight species such as cysteine, cysteamine, and coenzyme A. GSH is usually less than the total cellular NPSH, and with thiol reactive agents, such as diethyl maleate (DEM), its rate of depletion is in part dependent upon the cellular capacity for its resynthesis. If resynthesis is blocked by buthionine-S,R-sulfoximine(BSO), the NPSH, including GSH, is depleted more rapidly, Cellular thiol depletion by diamide, N-ethylmaleimide, and BSO may render oxygenated cells more sensitive to radiation. These cells may or may not show a reduction in the oxygen enhancement ratio (OER). Human A549 lung carcinoma cells depleted of their NPSH either by prolonged culture or by BSO treatment do not show a reduced OER but do show increased aerobic responses to radiation. Some nitroheterocyclic radiosensitizing drugs also deplete cellular thiols under aerobic conditions. Such reactivity may be the reason that they show anomalous radiation sensitization (i.e., better than predicted on the basis of electron affinity). Other nitrocompounds, such as misonidazole, are activated under hypoxic conditions to radical intermediates. When cellular thiols are depleted peroxide is formed. Under hypoxic conditions thiols are depleted because metabolically reduced intermediates react with GSH instead of oxygen. Thiol depletion, under hypoxic conditions, may be the reason that misonidazole and other nitrocompounds show an extra enhancement ratio with hypoxic cells. Thiol depletion by DEM or BSO alters the radiation response of hypoxic cells to misonidazole. In conclusion, we propose an altered thiol model which includes a mechanism for thiol involvement in the aerobic radiation response of cells. This mechanism involves both thiol-linked hydrogen donation to oxygen radical adducts to produce hydroperoxides followed by a GSH peroxidase-catalyzed reduction of the hydroperoxides to intermediates entering into metabolic pathways to produce the original molecule.

Animals↗
PubMed1982

Inhibition of glycolysis of mammalian cells by misonidazole and other radiosensitizing drugs. prevention by thiols.

Prolonged anaerobic incubation of Ehrlich ascites tumor cells and Chinese hamster V79-379A cells with misonidazole, desmethylmisonidazole, or niridazole led to inhibition of both glucose consumption and lactate formation. This effect was measured in cells washed free of the nitro compounds and resuspended in fresh buffer or medium. The degree of inhibition of glucose utilization was related to drug concentration, and to the rate of metabolic reduction, as measured under aerobic conditions by KCN-insensitive oxygen consumption. Misonidazole-induced inhibition of glycolysis developed concurrently with depletion of intracellular non-protein thiol (NPSH) and was protected against by the presence of cysteamine, cysteine and, to some extent, GSH in the cell incubate. These findings suggest diethyl maleate was used to deplete 90% of the endogenous NPSH, but this depletion did not alter the effects of misonidazole on glycolysis.

Anaerobiosis↗
PubMed1979

Interactions of the carcinogen 4-nitroquinoline 1-oxide with the non-protein thiols of mammalian cells.

The carcinogen 4-nitroquinoline 1-oxide (4-NQO) was found to rapidly deplete non-protein thiols (NPSH) from Ehrlich ascites tumor cells and V79 Chinese hamster fibroblasts. The effects of NPSH on 4-NQO metabolism were studied by measuring 4-hydroxyaminoquinoline 1-oxide formation, CN- -insensitive oxygen consumption, and reduction of ferricytochromes c + c1 in normal cells and in cells pretreated with the thiol reagent N-ethylmaleimide. Removal of thiols before treatment with 4-NQO resulted in increased production of 4-hydroxyaminoquinoline 1-oxide and increased production of nitro radicals. The NPSH thus appeared to play a significant role in 4-NQO detoxification. Glutathione, when present in culture medium during 4-NQO treatment, protected V79 cells from 4-NQO toxicity. Several mechanisms for reaction of 4-NQO with intracellular NPSH were indicated. Both V79 and Ehrlich cells contained appreciable amounts of glutathione S-transferase (EC 2.5.1.18), which catalyzes the nucleophilic substitution of the nitro group of 4-NQO with thiols. Greater thiol loss under oxic than under hypoxic conditions suggested oxidation by superoxide, peroxide, or hydroxyl radical formed in the course of 4-NQO reduction. In addition, reaction of thiols with nitro radicals or with nitrosoquinoline 1-oxide was indicated by the inhibitory effect of glutathione on oxygen consumption in solutions of 4-NQO and sodium ascorbate.

4-Nitroquinoline-1-oxide↗
PubMed1977

Metabolism of benzo(a)pyrene by guinea pig pancreatic microsomes.

The in vitro microsomal metabolism of the strain 13 guinea pig pancreas was investigated by determining the benzo(a)pyrene (BP) hydroxylase activity in the 9000 x g supernatant and microsomal pellet. BP hydroxylase activity in both 9000 x g supernatant and microsomal pellet of the pancreas was less than 1% of the activity in the respective liver fractions, However, pretreatment of animals with methylcholanthrene or BP at 20 mg/kg, for either 1 day or 3 consecutive days, markedly enhanced the BP hydroxylase activity of pancreatic microsomes over that of controls; the induction in the liver microsomes was less than 2-fold over that of controls. The hydroxylation of BP by pancreatic microsomes was linear with time over a 30-min period, with the rate of hydroxylation dependent on both the enzyme and substrate concentrations.

Animals↗
PubMedDate not supplied

Misonidazole-induced biochemical alterations of mammalian cells: effects on glycolysis.

Incubation of Ehrlich ascites tumor cells with misonidazole under aerobic conditions causes a stimulation of glucose consumption which is probably related to stimulation of hexose monophosphate shunt activity. Incubation of Ehrlich cells with 5 mM misonidazole under hypoxic conditions results in a time-dependent inhibition of glycolysis, seen when treated cells are washed and resuspended in fresh buffered saline or media. This inhibition does not appear to be related to breakdown and loss of pyridine nucleotides from the cells during misonidazole treatment, nor is it a consequence of cell death. Post-incubation in buffer containing cysteine or cysteamine restores cellular glycolytic activity to near control levels. Partial inhibition of glucose consumption after incubation with misonidazole also occurs with V79-379A, V79-171B, EMT6 and A549 cell lines. The extent of inhibition varies among the lines, but is accompanied by approximately a 50% reduction in intracellular non-protein thiol levels in all cases. Ehrlich cells incubated anaerobically with misonidazole lose their response to the uncoupler 2,4-dinitrophenol, which normally increases glucose consumption, and exhibit less ability to metabolize pyruvate. Cells incubated under anaerobic conditions but in the absence of misonidazole do not show these effects.

Aerobiosis↗
PubMedDate not supplied

A biochemical basis for the radiosensitizing and cytotoxic effects of chlorpromazine hydrochloride in vitro and in vivo.

There have been many investigations on the radiosensitizing and cytotoxic effects of chlorpromazine, both in vitro and in vivo. We have extended the studies to include the chemotherapeutic effect of chlorpromazine on lymphosarcoma and on a mammary adenocarcinoma arising spontaneously in breeding female CBA mice. Chlorpromazine was ineffective in the former tumor but enhanced the longevity of tumor-bearing mice in the latter case. The drug was also observed to potentiate the hyperthermic killing of L5178Y cells and inhibit cellular oxygen utilization by cultured mammalian cells. The possible mechanisms involved are discussed.

Animals↗
PubMedDate not supplied

Enhanced cytotoxicity of melphalan by prolonged exposure to nitroimidazoles: the role of endogenous thiols.

It was first demonstrated that prolonged exposure of hypoxic V-79 cells to misonidazole prior to irradiation produced an increased radiosensitization in 1977; it was postulated that the reduction of misonidazole resulted in intermediates capable of depleting cells of endogenous thiols, substances known to play a role in the hydrogen repair of target radicals produced by ionizing radiation. The present study shows that a prolonged exposure of V-79 cells to a variety of nitroimidazoles (misonidazole, Ro-05-9963, SR-2508, and MTR1-80) results in an enhanced cytotoxicity when these cells are subsequently exposed to melphalan. This process of enhanced melphalan toxicity occurred only when cells were pretreated with misonidazole under hypoxic conditions, suggesting that nitroreduction is necessary for chemosensitization as it is for increased radiosensitization. Different nitroimidazoles tested vary in the extent to which they sensitize cells to the subsequent action of melphalan. Repair from a misonidazole pretreatment is essentially complete by six hours. This study demonstrated that cysteamine could reduce the cytotoxicity of misonidazole and the enhancement of melphalan toxicity. This was an effect reversible with time and one implying similar mechanisms for the preincubation effect observed in vitro for radiation and chemotherapy agents.

Animals↗