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C J Koch

Publications and source records attributed to C J Koch.

At least 109 records · Page 6Linked to original sources
PubMed1988

Oxygen dependence of product formation in irradiated adenosine 5'-monophosphate.

The formation of (R)- and (S)-8,5'-cycloadenosine 5'-monophosphate (8,5'-cycloAMP), 8-hydroxyadenosine 5'-monophosphate (8-hydroxyAMP), and radiolytic adenine release from irradiated solutions of adenosine 5'-monophosphate (5'-AMP) was measured as a function of increasing liquid-phase oxygen concentration. Three classes of specific molecular damage were identified on the basis of the oxygen dependence for product formation. Major changes in product yield occurred near the range of oxygen concentrations associated with the radiobiological oxygen effect. In addition to these data, systematic increases in the concentration of hydrogen peroxide at the time of irradiation resulted in an increase in the yield of 8-hydroxyAMP and a component of radiolytic adenine release in nitrogen-saturated solutions of 5'-AMP. However, no changes in the yield of the 8,5'-cyclonucleotides were observed under these conditions.

Adenine↗
PubMed1987

Is misonidazole binding to mouse tissues a measure of cellular pO2?

Misonidazole (MISO), a hypoxic cell radiosensitizer, forms covalently-linked adducts to cellular molecules as a result of bioreductive metabolism, a process which is strongly dependent upon oxygen concentration. MISO binding to liver tissue taken from air-breathing mice was three to five times greater than binding to other normal tissues. The relative binding of [14C]MISO to various mouse tissue cubes in vitro was measured by autoradiography as a function of defined oxygen concentrations, and standard curves (binding rate vs oxygen concentration) were generated. The oxygen concentration for half-maximum binding as well as the maximum and minimum binding rates (grains per 100 micron 2) observed for liver tissue were not significantly different from those measured for brain or heart tissue. These results, along with previously published data on MISO binding to isolated hepatocytes in vitro, suggest that the elevated binding to liver in vivo may result, in part, from the organ existing at a significantly lower pO2 than other normal tissues. They also suggest that this drug adduct procedure could be developed as a sensitive method for the quantitative measurement of tissue pO2 at the cellular level.

Animals↗
PubMed1987

Oxygen dependence of binding of misonidazole to rodent and human tumors in vitro.

Misonidazole, a 2-nitroimidazole, has been shown to form metabolically induced adducts to cellular molecules at a very high rate in the absence of oxygen, and this rate decreases substantially as the oxygen concentration increases. Thus, it has considerable potential as a marker for hypoxic, radiation resistant cells in tumors. The dependence of the rate of adduct formation (binding) on oxygen concentration was studied for EMT6/Ed, Walker 256, and Dunning R3327-AT rodent tumors and for two human colon carcinomas, a human melanoma, and a human breast carcinoma. Fragments of these tumors were incubated with [14C]- or [3H]misonidazole in vitro at several oxygen concentrations and the quantity of misonidazole bound was determined from autoradiographs as a function of distance from the surfaces of the fragments. The Km of binding inhibition (oxygen concentration for half-maximal binding) for the tumors varied by a factor of 10. The range was centered on the range of values reported for the Km of cellular radioresistance (oxygen concentration for half-maximal radioresistance). The patterns of binding at depth within the tumor fragments indicated that gradients of cellular waste products and nutrients other than oxygen had minimal effects on binding. All tumors were capable of metabolizing oxygen to levels sufficiently low to yield the maximal binding rate, but the distance of penetration of oxygen varied, indicating a range of at least 4 in rates of oxygen consumption. The ratio of misonidazole bound by stromal tissue versus tumor cells ranged from 0.9 for a colon tumor to 0.3 for a breast tumor. These properties of misonidazole binding indicate that it should be a good marker for radiobiological hypoxia in tumors, providing adequate controls can be performed.

Adenocarcinoma↗
PubMed1987

Fluorescence immunohistochemical detection of hypoxic cells in spheroids and tumours.

Polyclonal antibodies have been raised in rabbits to a haemocyanin adduct of a reductively-activated, fluorinated analogue of misonidazole. Fluorescence immunohistochemical studies show that the polyclonal antibodies bind to spheroid sections and tumour sections in patterns similar to those revealed by autoradiographic studies with a tritium-labelled derivative of the fluorinated misonidazole analogue.

Animals↗
PubMed1986

Combined radiation-protective and radiation-sensitizing agents. III: Radiosensitization by misonidazole as a function of concentrations of endogenous glutathione or exogenous thiols.

Radiosensitization of V79 Chinese hamster fibroblasts by 0.5 mM misonidazole is a smooth function of endogenous glutathione (GSH) levels as modulated upwards by pre-incubation in medium containing cysteamine, or downwards by pre-incubation in medium containing buthionine sulfoximine. The enhancement ratio (radiation sensitivity in nitrogen/radiation sensitivity in nitrogen +/- sensitizer or thiol) varies from 1.3 at 12 mM to 2.25 at less than 0.1 mM endogenous GSH. The enhanced radiosensitivity of thiol-depleted hypoxic cells is reversed when exogenous thiols are added, and for equivalent ER, the exogenous thiol concentrations are much lower than the endogenous GSH concentrations. Measurement of intracellular drug concentrations amplified rather than diminished the above discrepancy, since intracellular concentrations of cysteamine were lower and glutathione much lower than the extracellular concentrations. Three possible explanations are addressed: an external membrane component of damage is involved, long-range protection to DNA target radicals is possible from outside the cell (e.g., donation of electrons), and (c) endogenous glutathione is not in a free or exchangeable state (e.g., bound).

Animals↗
PubMed1986

Binding of 3H-misonidazole to solid human tumors as a measure of tumor hypoxia.

Treatment-resistant, chronically hypoxic tumor cells have been assumed to exist in some solid human tumors, limiting their curability. To date, six patients with different types of tumors have been studied using radioactive labelled electron affinic compounds that bind to hypoxic cells. Although the gross clinical appearance of the tumors in all six patients was of a large and fixed mass which might on clinical grounds be expected to contain hypoxic cells, we have observed drug binding to hypoxic regions in only two, a rapidly growing small cell lung cancer (SCLC) and a malignant melanoma. The hypoxic fraction of the malignant melanoma was found to be 6% and the SCLC tumor approximately 10%. We have observed that areas of maximum adduct formation can be found in tumor cells immediately adjacent to blood vessels, suggesting that blood flow over the labelling interval was restricted. These preliminary studies suggest that sensitizer adduct formation in human tumor tissue may be a useful measure of tissue pO2 at the cellular level and that tumor hypoxia might be more related to the rate of tumor growth and histological grading than to tumor size.

Autoradiography↗
PubMed1986

A novel technique for measuring human tissue pO2 at the cellular level.

Some electron-affinic drugs, developed as hypoxic cell radiosensitizers, become selectively bound to the molecules of hypoxic cells by metabolism. This technique has been used to identify zones of chronically hypoxic cells in multicellular spheroids and animal tumours. Tritiated-misonidazole was administered to a patient with advanced melanoma 22 h prior to the surgical resection of a large metastatic s.c. lesion growing on the face. Autoradiographic analysis of histological sections revealed zones of intense labelling by the radioactive drug, indicative of tumour cells which were chronically hypoxic. This technique appears to provide an indirect measurement of tissue pO2 at the cellular level from which estimates of the tumour hypoxic fraction can be made. These data are encouraging as regards the development of 'sensitizer-adduct' procedures for the invasive and non-invasive measurement of hypoxia in both tumours and normal tissues.

Adult↗
PubMed1986

Measurement of low levels of oxygen and their effect on respiration in cell suspensions maintained in an open system.

The presence of low levels of oxygen may have profound effects on the cytotoxic activity of radiation, radiosensitizers, and bioreductive alkylating agents. As others have shown, low oxygen tensions may significantly alter rates of cellular and chemical oxygen consumption. When experiments are performed at very low oxygen concentrations, the opposing effects of oxygen leakage into and cellular/chemical oxygen consumption from the system can lead to unpredictable results. Use of a newly designed, highly sensitive Clark-type oxygen sensor has permitted accurate and reproducible measurement of low levels of oxygen. Cellular depletion of oxygen at various cell densities has been monitored for a series of oxygen tensions in solution and the corresponding respiration rates have been calculated. Although oxygen depletion was found to be quite significant at low oxygen tensions, not all oxygen present could be removed by cellular respiration. Respiration rate decreased as oxygen tension decreased and approached zero at low oxygen tensions. This result was independent of cell density. A model is presented to account for the observed effect of oxygen tension on cellular oxygen utilization.

Animals↗
PubMed1985

Binding of misonidazole to hypoxic cells in monolayer and spheroid culture: evidence that a side-chain label is bound as efficiently as a ring label.

The binding of ring-labelled and side-chain labelled misonidazole to hypoxic cells in monolayer and spheroid cultures of mammalian cells has been compared. The kinetics and patterns of binding for the two labelled compounds are indistinguishable. This finding has implications for the mechanism of binding and for the design of misonidazole analogues which might be used to identify hypoxic zones in tumours.

Animals↗
PubMed1985

The influence of oxygenation on the 19F spin-lattice relaxation rates of fluosol-DA.

The spin-lattice relaxation rates (1/T1) were measured at 94.1 MHz for six peaks in the 19F NMR spectrum of the perfluorochemical blood substitute fluosol-DA, which contains a mixed emulsion of perfluorodecalin and perfluorotripropylamine. Each of these rates increased linearly with the percentage of oxygen dissolved in the emulsion. Relative values of the linear increase for different peaks established that, for perfluorotripropylamine in the mixed emulsion, the oxygen-fluorocarbon interaction is loosely but preferentially oriented in a manner similar to that previously established for other pure fluorocarbons. The uncertainty in the oxygen level estimated from T1 measurements is somewhat less than 5% O2 and it is thus established that quantitative non-invasive oxygenation measurements can be made to sufficient precision by this approach, using fluosol-DA and 19F spin-lattice relaxation.

Contrast Media↗
PubMed1984

Metabolism induced binding of 14C-misonidazole to hypoxic cells: kinetic dependence on oxygen concentration and misonidazole concentration.

Under conditions of extreme hypoxia, metabolic products of the metabolism of misonidazole bind to cellular molecules at a rate which is linear with time and proportional to the square root of misonidazole concentration. Very small amounts of oxygen reduce the overall rate of binding and cause a change in the dependence on misonidazole concentration from square root (half order) to linear (first order). Because of the known electron affinity of misonidazole, a model is presented whereby the nitro-group is reduced to a radical in a first order reaction. This radical binds to cellular molecules in a slow first order reaction and either disproportionates or dimerizes in a fast second order reaction. Based on the overall effect of oxygen on the kinetics of the rate of binding, the radical is tentatively assumed to be the 3 electron reduction product.

Animals↗
PubMed1984

Binding of misonidazole to V79 spheroids and fragments of Dunning rat prostatic and human colon carcinomas in vitro: diffusion of oxygen and reactive metabolites.

Differences were noted previously in the binding of 14C-Misonidazole (MISO) to V79 and EMT6 spheroids when incubated at low oxygen levels. While binding to the viable cells in EMT6 spheroids was uniform, in V79 spheroids the binding rate increased gradually with distance from the spheroid surface. Further data reported here indicate that the Km for the inhibition of binding by oxygen is lower in V79 than EMT6 spheroids, so that part of the non-uniformity of binding to V79 spheroids can be explained by diffusion of small amounts of oxygen through the entire rim of viable cells. Diffusion of reactive metabolites of MISO out of the spheroid previously was considered an unlikely explanation. Further evidence to support this interpretation is presented here. Patterns of binding of 3H-MISO to Dunning and human colon carcinomas are presented which are consistent with the interpretation that most of the reactive metabolites are confined to the cell in which they are produced. This conclusion is based on a substantial difference in the rate of binding to adjacent stromal and tumor elements.

Animals↗
PubMed1984

A thin-film culturing technique allowing rapid gas-liquid equilibration (6 sec) with no toxicity to mammalian cells.

A method is described for inoculating mammalian cells onto the central area of glass petri dishes. The medium depth above the cells is only 100 microns for an added medium volume of 1 ml and increases linearly and rapidly with additional medium. The theoretical time constant for equilibration of the medium with the gas is related to the square of the medium depth. The experimental time constant was measured in two different ways for large and small medium depths, giving excellent agreement with the theoretical values. Although the time constant is only 6 sec for the case of 1 ml of added medium, there is no drying out of the medium or toxicity to the cells because of a large reservoir of medium in the meniscus at the periphery of the dish.

Animals↗
PubMed1984

The effect on the Km for radiosensitization at 0 degree C of thiol depletion by diethylmaleate pretreatment: quantitative differences found using the radiation sensitizing agent misonidazole or oxygen.

Pretreatment of V79- WNRE cells with 150 microM diethylmaleate for 1 hr at 37 degrees C caused a decrease in intracellular glutathione levels to approximately 10-15% of control levels (0.5 vs 5.0 nmol/10(6) cells). The cells could be washed free of diethylmaleate and held at 0 degree C for several hours without toxicity and with no increase in glutathione concentration, although the glutathione concentration rapidly increased to normal levels at higher temperatures. Survival curves were determined as a function of oxygen or misonidazole concentration (the latter in the absence of oxygen). A new "thin-film" technique was used to avoid changes in oxygen concentration because of radiochemical or cellular oxygen consumption. Glutathione depletion itself caused a small but consistent radiosensitization of hypoxic cells (dose enhancement ratio of 1.2). However, glutathione depletion caused a profound change in the radiosensitizing efficiency of misonidazole, with a decrease in Km of about sevenfold from 0.6 to 0.09 mM. In contrast, only a 2.5-fold decrease was found in the Km for radiosensitization by oxygen with diethylmaleate pretreatment. These results suggest a fundamental problem with the conventional theory of radiosensitivity whereby one considers a first-order competition for reaction with target radicals between radical-fixing versus radical-repairing species. It also suggests difficulties in the interpretation of glutathione as the only endogenous protective species.

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↗