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Biomedical subjects

W R Inch

Publications and source records attributed to W R Inch.

At least 19 recordsLinked to original sources
PubMed1990

HPLC and flow cytometric analyses of uptake of adriamycin and menogaril by monolayers and multicell spheroids.

We have used both HPLC and flow cytometry to measure and compare the uptake of two anthracyclines, menogaril (MEN) and Adriamycin (ADR), in V79 Chinese hamster lung fibroblasts grown as monolayers and as 650 microns multicell spheroids. In order to compare intracellular drug accumulation in spheroid cells measured by the two methods, we converted mean channel fluorescence of the flow cytometer to drug uptake expressed as ng/10(6) cells by using a standard curve. The standard curve related the flow cytometric mean channel fluorescence, of monolayer cells exposed to either drug, to the intracellular drug accumulation determined by HPLC. This standard curve was then used to convert the mean channel fluorescence of cells from drug-exposed spheroids to ng/10(6) cells. Our results show that equal intracellular drug accumulation (determined by HPLC) in spheroids and monolayers does not result in equal cellular fluorescence emission (determined by flow cytometry) by these 2 cell populations. For example, monolayer cells with an intracellular MEN accumulation of 650 ng/10(6) cells, emit 40 units of fluorescence as measured by flow cytometry. However, spheroid cells with the same intracellular accumulation emit about 80 units of fluorescence. This results in the intracellular MEN uptake in spheroids measured by flow cytometry being as much as 2- to 3-fold higher than that measured by HPLC. Intracellular ADR accumulation measured by flow cytometry was also higher than that obtained by HPLC. In spite of the quantitative difference between the two methods, qualitatively both methods gave similar results. Thus, both techniques showed that at equal drug concentration in medium drug uptake in monolayers was much greater than in spheroids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Radioprotective effect of free radical scavenging enzymes.

Ionizing radiation is an important treatment modality in the management of head and neck squamous cell carcinoma. The superoxide (O2-) and hydroxyl (OH.) radicals produced from oxygen and the radio-hydrolysis of water are responsible for most of the DNA and lipid membrane injury caused by radiotherapy. Superoxide dismutase (SOD) and catalase (CAT) are intracellular enzymes that scavenge the superoxide and hydroxyl radicals respectively. The effect of intravenous SOD and CAT on acute and delayed radiation injury was investigated in a rat model. Catalase was shown to reduce the severity of radiation-induced changes in both the vascular endothelium and squamous epithelium. SOD, alone or with CAT, showed no radioprotective effect. As intravenous catalase does not penetrate intracellularly it should have no effect on the tumoricidal effect of radiation. Further investigation of catalase as an agent to reduce the acute side-effects of radiotherapy is warranted.

Animals↗
PubMed1989

51Cr release and oxidative stress in the lens.

Examination of the opaque areas of human cortical cataracts has shown that a large portion of the opacity could be attributed to the globules found there. We tested models involving globule formation as a result of oxidative damage to rat lens cells in culture and whole chick embryo lenses. When cell monolayers from a lens cell line were exposed to oxidizing conditions they developed globules on the cell surface. The cells were protected from damage by the addition of glutathione and vitamin C. Thirteen-day chick embryo lenses were also incubated in oxidizing conditions and the amount of cellular damage was assessed using a chromium-51 release assay we have developed. After 24 hr the percent 51Cr in the medium increased by an average of 20% as a result of 10 mM hydrogen peroxide treatment. The addition of the 10 mM vitamin C to the hydrogen peroxide significantly reduced the 51Cr leakage to the control level. Light microscopy of sections of the lens showed a breakdown of the equatorial fibre arrangement in the presence of H2O2, while addition of vitamin C restored the fibre organization to almost normal. The findings suggest that oxidative stress is an important step in cataractogenesis and point towards the use of water soluble antioxidants as protective agents.

Animals↗
PubMed1987

Resistance of V79 multicell spheroids to mitoxantrone: drug uptake and cytotoxicity.

We have previously shown that V79 multicell spheroids are resistant to the anthracenedione mitoxantrone (1). In this paper we demonstrate that this resistance is not solely due to restricted drug penetration into the spheroid, but also to an altered intrinsic resistance of the cells when grown as a three-dimensional structure. We have studied the uptake and toxicity of mitoxantrone in V79-OCF4 monolayers, 100 micron spheroids, 650 micron spheroids, and outer and inner cells of 650 micron spheroids. The LD90 for cells exposed as monolayers to mitoxantrone for two hours was 0.016 microgram/ml, 0.055 microgram/ml for 100 micron spheroids, 1.5 micrograms/ml for outer spheroid cells and 6.2 micrograms/ml for inner spheroid cells. Uptake of [14C]mitoxantrone was linear for all populations with no plateau up to the highest doses used. The uptake of drug required to kill 90% of the cells in a population (UP90) of monolayers was 3.7 ng/10(6) cells, 10.7 ng/10(6) cells for 100 micron spheroids, 169 ng/10(6) cells for outer spheroid cells, and 146 ng/10(6) cells for inner spheroid cells. The relative resistance of spheroids compared to monolayers, based on drug concentration in the medium, was 3.4 for small spheroids, 92 for outer cells, and 390 for inner cells. When cell survival was normalized to drug uptake, the relative resistance of spheroids to monolayers was 2.9 for small spheroids, 46 for outer cells and 40 for inner cells of large spheroids. The data demonstrate that the resistance of multicell spheroids to mitoxantrone is not solely due to restricted drug penetration into the cell aggregate but is also due to a spheroid-induced altered intrinsic resistance of the V79 cells.

Cell Survival↗
PubMed1986

Modeling of proton spin relaxation in muscle tissue using nuclear magnetic resonance spin grouping and exchange analysis.

NMR spin relaxation experiments performed on healthy mouse muscle tissue at 40 MHz and 293 K are reported. The spin-lattice relaxation experiments were performed using different combinations of selective and nonselective radio frequency pulses. Relaxation experiments in the rotating frame at H1 = 10, 5 and 1 G are also reported. The experimental results were analyzed using the spin-grouping method, which yields the sizes of the resolved magnetization components as well as their T2's and T1's (or T1p's) for the nonexponential relaxation functions. These results were analyzed further for the exchange between different spin groups. It has been found that to explain all of these experimental data it was necessary to use a four-compartment model of the muscle tissue that consists of a lipid spin group, a "solid-like" spin group (mainly proteins), a "bulk water" spin group and a "bound water" spin group. The chemical exchange rate between "bulk" and "bound" water was found to be 29 +/- 9s-1 at room temperature. The exchange rate between the bound water and the solid moderator was estimated to be approximately 500 s-1.

Animals↗
PubMed1986

Exercise muscle metabolism measured by magnetic resonance spectroscopy.

Nuclear magnetic resonance imaging (MRI) is a technique which gives body images in much the same way as X-ray tomography. Most images are produced from signals of mobile hydrogen atoms in the tissues. Since bone contains few such atoms the images do not show bone material but allow marrow to be observed. Recently the technique has been extended to include spectroscopy of atoms of 31P, 13C and several others important in cell and tissue metabolism. The technique which uses a radiofrequency surface coil placed over the tissue of interest, is called topical magnetic resonance spectroscopy (MRS). Magnetic resonance spectroscopy which measures phosphorus nuclei can be used to study phosphorus in muscle. The same instrument can be used to study hydrogen density distribution. It can monitor muscle metabolism during exercise and muscle energetics can thus be evaluated. MRS gives a more accurate picture of the in vivo proportions of high energy phosphates than conventional biochemical analysis, which requires muscle biopsy, it can be used to monitor living tissue in a non-destructive way.

Aerobiosis↗
PubMed1985

Changes in ultrastructure and function of hypoxic V79 fibroblast cells after treatment with misonidazole.

Morphologic and enzymatic changes due to exposure to the radiosensitizing chemical, misonidazole, have been identified in V79 cells grown in a system in which oxygen tensions and culture density have been controlled. Misonidazole prevented the increase in mitochondrial size normally seen during exposure of these cells to conditions of moderate hypoxia (2 X 10(3) ppm O2). Mitochondrial size was also significantly decreased in cells from exponential cultures exposed to 1 mmol/l misonidazole. Morphologic changes to the mitochondria that varied from data reported elsewhere were also noted. Misonidazole caused a significant initial decrease in cytochrome oxidase activity after 4 hours of exposure of aerobic and moderately hypoxic cultures that did not return to normal in chronically hypoxic cells during continued exposure to the drug.

Animals↗
PubMed1985

Characterization of normal and malignant mouse tissue by NMR lineshape-relaxation correlations in the rotation frame.

With the NMR lineshape-relaxation correlation technique the relaxation times and the relative magnitudes of the proton spin groups characterized by these respective relaxation times are obtained. In most tissues the spin-lattice relaxation in the rotating frame is nonexponential. The magnetization evolution can be represented as the sum of three exponential decays characterized by three T1rho relaxation times. The short T1rho component, corresponding to the rigid network protons, gives the most interesting information on the tissue. Results at 38 MHz are given for muscle, spleen, lung, and tumor EMT6 fibrosarcoma grown in the dorsal subcutaneous tissue of BALB/c mice. The absence of the magnetization component with short T1rho and short T2 in spleen tissue of tumorous mice demonstrates that the spleen is swollen to at least 5 times its original size so that the relative magnitude of the magnetization corresponding to collagen network is proportionally reduced. The NMR correlation technique is thus shown to be appropriate for such quantitative analysis.

Animals↗
PubMed1985

Ultrastructural changes in V79 hamster lung fibroblasts during hypoxic exposure.

Cells in tumors that are deprived of their blood supply become hypoxic. These stressed cells adapt to their new environments by altering their metabolic regimen which in time induces cellular structure changes. The morphologic make-up of these O2-deprived cells is the focal point of this electron microscopy study. V-79 hamster lung fibroblast cells grown as monolayer cultures were examined under controlled culture density and oxygen tensions - normal aerobia (2.1 X 10(5) ppm O2), and extreme hypoxia (less than 10 ppm O2). Electron micrographs of these cells demonstrated a loss of structural mitochondrial integrity accompanied with large increases in both mitochondrial and lipid vacuole size following exposure to extreme hypoxia. When these cells were reoxygenated, those mitochondria which had not become degenerate returned to their normal state however, lipids still continued to accumulate in vacuoles for a further 6 h. Addition of 1 mM palmitic acid to aerobic cultures evoked similar lipid and mitochondrial irregularities as were observed in hypoxic cells although, the latter were not as marked. When this saturated fatty acid was added to hypoxic cells no further structural alterations were seen. The cellular changes manifested during this study were subjected to quantitative measurements and these results have given an insight into the scope and variety of ultrastructural changes which have resulted from exposure of cultured cells to hypoxic conditions.

Animals↗
PubMed1983

Radiation cataract formation diminished by vitamin E in rat lenses in vitro.

Eye lens cataract is a late effect of exposure to ionizing radiation. Depending on the dose and quality of radiation impinging on the lens, the development of a clinically discernible cataract usually takes several months in animal models, when the lens is irradiated in situ. However, we have developed a rapid in vitro assay with the isolated intact rat lens to study the effect of radiation and influence of antioxidants as protective agents. After only 24 hr of post-irradiation incubation at 35.5 degrees C in complete medium 199 + 10% foetal calf serum, damage in the form of globular degeneration subcapsularly and 'holes' in the cortical fibre cells is detectable. Doses as low as 0.10 Gy seem to be capable of causing some damage, and vitamin E (2.4 microns) in the medium confers some protection to the irradiated lens.

Animals↗
PubMed1982

Improved characterization of healthy and malignant tissue by NMR line-shape relaxation correlations.

We performed a relaxation-line-shape correlation NMR experiment on muscle, liver, kidney, and spleen tissues of healthy mice and of mouse tumor tissue. In each tissue studied, five spin groups were resolved and characterized by their relaxation parameters. We report a previously uncharacterized semi-solid spin group and discuss briefly the value of this method for the identification of malignant tissues.

Animals↗
PubMed1980

Buoyant density of EMT6 fibrosarcoma cells: time course of the density changes after growth in hypoxia.

EMT6 fibrosarcoma cells were grown to the exponential phase in tissue culture and incubated at 37 degrees C under hypoxic conditions. Buoyant density was determined as a function of the time in hypoxia. Hypoxia was produced in two ways. The first involved incubation of the cells in sealed aluminum chambers containing 95% N2, 5% CO2 gas, and < 10 ppm oxygen, resulting in the cells rapidly becoming exposed to the hypoxic environment. After incubation at 37 degrees C, they were centrifuged in linear Ficoll gradients to their isopycnic density. A significant decrease in density was found after 4 h, and prolonged incubation up to 24 h did not result in further change. This density change was reversible on transfer back to aerobic conditions, with the hypoxic cells reverting to their aerobic density after about 10 h reincubation in air. The second method of producing hypoxia involved growing about 8 X 10(6) cells in a medium-filled air-tight container. Hypoxia was produced gradually as the oxygen in the medium was consumed by cellular respiration. Similar results were obtained; that is, hypoxic cells became significantly less dense. However, when the level of hypoxia was varied between 4000 and < 10 ppm at 2-h intervals after the cells had depleted all of the original oxygen, no significant difference in density was found between hypoxic and aerobic cells.

Animals↗
PubMed1979

Cytotoxicity of glucose analogues in V79 multicell spheroids.

2-Deoxy-D-glucose (2DG) and 5-thio-D-glucose (5TG) are glucose antimetabolites that are known to be selectively toxic to hypoxic cells grown as single cells or as monolayer cultures. These analogues were toxic to Chinese hamster V79 cells grown as multicell spheroids even under aerobic conditions. When spheroids, 500- to 600-microns diameter, were exposed to 7.5 mM of these chemicals for 3 days, the number of clonogenic cells per spheroid dropped to 50% for 5-thio-D-glucose and 20% for 2-deoxy-D-glucose, relative to control values. Survivals were reduced to less than 1% when the experiment was repeated in glucose-free medium. Scanning electron photomicrographs of spheroids treated with 7.5 mM of either analogue showed extensive damage to the outer cells. The cell killing observed was much more than could be predicted on the basis of the hypoxic fraction known to be present in these spheroids. The crowded tumor-like environment may make the cells vulnerable to the cytotoxic action of glucose analogues and other glycolytic inhibitors.

Animals↗
PubMed1979

Proton T1 study of coverage parameter changes in tissues from tumor-bearing mice.

measurements of water proton spin-lattice relaxation time, T1, at 20 and -15 degrees C have been performed in spleen, kidney, liver, and muscle tissues from tumor-bearing mice, as well as in tumors grown in their dorsal subcutaneous tissues. All mice used were either from the C3H/HeJ or BALB/c strain. At - 15 degrees C the T1's of tissues of a given type from tumor-bearing and healthy mice are essentially the same. It is shown that in spleen the increased T1 from tumor-bearing mice can only be explained in terms of a large change in the water coverage parameter of macromolecules. In liver, muscle, and tumors the increased water content accounts for the changes in T1, while kidney represents an intermediate case.

Animals↗
PubMed1978

Comparison of the densities of clonogenic cells from EMT6 fibrosarcoma monolayer cultures, multicell spheroids, and solid tumors in ficoll density gradients.

The densities of viable EMT6 cells grown in vitro as monolayer exponential and plateau-phase cells or as multicell spheroids or in vivo as a solid tumor in BALB/c mice were determined with the use of isopyknic centrifugation in linear Ficoll gradients. Exponential cells banded at a density of 1.069 g/ml, whereas plateau-phase cells appeared at 1.073 g/ml. Cells grown as spheroids were more dense than the monolayer cells and were recovered mainly at 1.081. Solid-tumor cells, separated under the same conditions, banded at 1.080. Narrowing the range of the Ficoll gradient failed to resolve more than one band of cells in the solid-tumor separation. This provides evidence that the density of cells obtained from the spheroids is greater than that of the monolayer cells but agrees well with the density of the solid-tumor cells. Ficoll was demonstrated to be nontoxic to the cells, and plating efficiency assays showed similar cell viabilities between noncentrifuged and centrifuged cells. The plating efficiency of the peak fraction of exponential cells after centrifugation was 68%, and that of plateau-phase cells was 50%. Corresponding figures for the multicell spheroid and solid-tumor cells were 62 and 28%, respectively. The recovery of cells after centrifugation in the Ficoll gradients ranged from 62 to 83%. The effects of cell load and centrifugation time on the density distributions of the EMT6 cells were also investigated.

Animals↗
PubMed1978

Cell survival in V79 multicell spheroids treated with dehydroascorbate, 5-thio-D-glucose and 2-deoxy-D-glucose.

We found that dehydroascorbic acid (DHA), 5-thio-D-glucose (5TG) and 2-deoxy-D-glucose (2DG) were toxic to monolayers of V79 cells under hypoxic conditions at 37 degree C but not at 0 degree C. Very little toxicity was observed at either temperature under aerobic conditions. When spheroids (600 micrometer diameter) were treated with 7.5 mM of these chemicals for periods up to 3 days, the number of viable cells per spheroid dropped to 70% for DHA, 50% for 5TG and 25% for 2DG relative to controls. These survivals were reduced to much lower values when the experiments were repeated in glucose-free medium.

Ascorbic Acid↗