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Biomedical subjects

J Hilton

Publications and source records attributed to J Hilton.

At least 91 records · Page 5Linked to original sources
PubMed1990

Direct demonstration of elevated aldehyde dehydrogenase in human hematopoietic progenitor cells.

Relative levels of cytoplasmic aldehyde dehydrogenase (ALDH) were determined in selected subpopulations of normal human bone marrow cells using a flow cytometric assay that simultaneously detects a cell surface antigen (as a marker of cell lineage and developmental stage) and the level of ALDH. The intracellular level of this enzyme has been shown to be directly related to cellular resistance to activated cyclophosphamide and is believed to be important in the survival of cells capable of repopulating marrow in autologous bone marrow transplant procedures. Western blot analysis and flow cytometric analysis of four murine cell lines with known ALDH levels were used to establish the relation between ALDH content and fluorescence with an affinity-purified anti-mouse ALDH antibody. An affinity purified anti-human ALDH antibody, characterized by immunoblotting of cytosolic extracts of cell lines with known ALDH content, was used to determine relative ALDH levels in the marrow subpopulations. We found that hematopoietic progenitor cells express the highest level of ALDH, while lymphocytes express the lowest level. Immature erythroid cells express ALDH at a level intermediate between progenitor cells and lymphocytes.

Aldehyde Dehydrogenase↗
PubMed1990

Glutathione protects cardiac and skeletal muscle from cyclophosphamide-induced toxicity.

Administration of cyclophosphamide at a dose which is lethal to 10% of control athymic nude mice resulted in sudden death within 3 h in all mice that had been pretreated with the glutathione synthesis inhibitor L-buthionine-SR-sulfoximine. In Fischer 344 rats pretreated with L-buthionine-SR-sulfoximine, the cyclophosphamide dose producing 100% acute toxicity was lowered from 500-150 mg/kg; cardiac monitoring revealed ventricular fibrillation to be the cause of death. These and additional studies reported demonstrate that cytoplasmic glutathione is an important protectant against the cardiac and skeletal muscle toxicity of cyclophosphamide and indicate that such toxicity may be substantially increased by glutathione depletion. Since diet and many drugs (including cyclophosphamide itself) are known to affect glutathione levels, the present studies suggest that cardiac and skeletal muscle glutathione content is likely to be a clinically significant determinant of the frequency and severity of the adverse drug interactions and systemic toxicity sometimes observed during cyclophosphamide therapy.

Animals↗
PubMed1990

Identification of contact allergens using the murine local lymph node assay: comparisons with the Buehler occluded patch test in guinea pigs.

A murine local lymph node assay has been developed for the identification of contact sensitizing chemicals. In the present study, the performance of the local lymph node assay has been evaluated with twenty-four coded chemicals of previously unknown skin sensitizing potential and the results compared with predictions made from concurrent occluded patch tests (Buehler tests) in guinea pigs. The data presented demonstrate that the local lymph node assay successfully identified those chemicals that were classified as moderate or strong skin sensitizers in the Buehler test. In the present series of experiments, chemicals predicted to be mild sensitizers in the Buehler test were classified as 'not strong sensitizers' in the local lymph node assay. In the majority of instances, the Buehler test and local lymph node assay were in agreement with regard to the identification of non-sensitizing chemicals. However, two chemicals that were classified as non-sensitizers in the guinea pig test exhibited positive responses in the local lymph node assay and were predicted to be sensitizers. Some coloured chemicals resulted in obscured Buehler readings and, here, assessment was based upon histological examination of the challenge site. These compounds were examined also in the local lymph node assay and similar predictions of sensitizing potential were made. Taken together, the data reveal close, but not absolute, concordance between the local lymph node assay and the Buehler test. The relative merits of these predictive test methods are discussed.

Allergens↗
PubMed1990

Contact sensitization of mice to nickel sulphate and potassium dichromate.

The ability of nickel sulphate and potassium dichromate to induce contact sensitization in mice has been investigated. Topical exposure of mice to 0.5% potassium dichromate in dimethylsulphoxide (DMSO) under occlusion resulted in specific contact sensitization in each of 7 experiments. Although sensitization to nickel sulphate under the same conditions was weak and somewhat variable, evidence for significant levels of contact allergy was obtained in 4 of 7 experiments. In addition, repeated topical application of both nickel sulphate and potassium dichromate in DMSO was found to result in the induction of lymphocyte proliferative responses in lymph nodes draining the site of exposure. These data indicate that epicutaneous exposure to both metal salts may cause specific contact sensitization in mice.

Animals↗
PubMed1990

Effect of superoxide dismutase in rabbits with peritumoral edema.

We evaluated the effect of SOD, an oxygen-free radical scavenger, on peritumoral edema in 20 rabbits. The VX2 carcinoma was transplanted to the brains of these New Zealand white rabbits. Detection of superoxide radicals in vitro was performed by incubating the VX2 tumor cells with NBT. For evaluation of the effect of SOD on the tumor cells, they were treated with free SOD or PEG-SOD before and after incubation with NBT. The animals were separated into three groups: group 1, control group; group 2, SOD-untreated tumor group; group 3, two SOD-treated groups-group 3a, treated with 10,000 U/kg PEG-SOD on day 1 and 4 after tumor transplantation and sacrificed on day 13; group 3b, treated with 10,000 U/kg PEG-SOD on day 7 and 10 and sacrificed on day 13. Brain edema was assessed by SG measurement. Our preliminary in vitro data indicated that the VX2 carcinoma produced superoxide radicals but that free SOD and PEG-SOD could not penetrate into tumor cells nor inhibit superoxide radicals. In vivo data also indicated that PEG-SOD failed to reduce peritumoral edema. It was concluded that intracellular uptake or penetration of SOD must first be achieved before any effect on peritumoral edema can be assessed.

Animals↗
PubMed1989

Increased melphalan activity in intracranial human medulloblastoma and glioma xenografts following buthionine sulfoximine-mediated glutathione depletion.

In previous studies we demonstrated that administration of buthionine sulfoximine (BSO) to athymic BALB/c mice bearing intracranial human glioma xenografts resulted in highly selective depletion of glutathione in neoplastic tissue with minimal effects on contralateral normal brain tissue. In the present study we treated athymic BALB/c mice bearing intracranial human glioma (D-54 MG) or medulloblastoma (TE-671) xenografts with melphalan alone or BSO followed by melphalan. Administration of BSO depleted intracellular glutathione to 7.5% of the control level. BSO plus melphalan resulted in a significant increase in median survival over that produced by melphalan alone: 45.3% versus 26.4% in TE-671 and 69% versus 27.6% in D-54 MG. These studies justify further efforts to modulate chemotherapeutic and radiotherapeutic interventions of primary malignant brain tumors by depletion of glutathione.

Animals↗
PubMed1989

Sampling strategies for water quality monitoring in lakes: the effect of sampling method.

Five different water sampling techniques have been compared in a series of lakes. In deep lakes, no significant differences were observed between mean summer nutrient concentrations measured in (i) a tube sample integrating over the photic zone taken from the deepest point; (ii) a surface dip sample taken at the deepest point; (iii) a surface dip sample taken by wading into the water's edge; (iv) a dip sample taken slightly further off shore by suspending a bottle below a buoy thrown out about 30 m from the shore; and (v) a sample taken along a short transect out from the shore using a model boat to transport the sample bottle. In shallower lakes the integrating tube sampler gave significantly higher estimates of mean concentrations than other methods due to the increase in volume of the unmixed hypolimnion which reduced the depth of the well mixed epilimnion to less than the tube length. For national survey purposes samples taken from the edge of the lake are the most cost effective.

Journal Article↗
PubMed1989

The murine local lymph node assay for identification of contact allergens: a preliminary evaluation of in situ measurement of lymphocyte proliferation.

A murine local lymph node assay for the identification of contact allergens has been developed. Contact sensitizing activity is measured as a function of lymphocyte proliferation in the draining lymph node following repeated application of the test agent to the dorsum of the ear. In original studies, lymphocyte proliferative activity was measured in vitro. In an attempt to remove the requirement for tissue culture, and thereby enhance the utility of the local lymph node assay as a predictive screening method, a new protocol has been evaluated in which proliferation in draining nodes is measured following i.v. injection of 3H-thymidine.

Allergens↗
PubMed1988

Buthionine sulfoximine-mediated depletion of glutathione in intracranial human glioma-derived xenografts.

D-54 MG, a human glioma-derived continuous cell line growing as subcutaneous or intracranial xenografts in athymic mice, was found to be sensitive to the effects of D,L-buthionine-(SR)-sulfoximine, a selective inhibitor of gamma-glutamylcysteine synthetase. Intraperitoneal administration of one dose of buthionine sulfoximine (BSO, 5 mmol/kg) resulted in depletion of total intracellular glutathione to 57 and 47% of control 12 hr, and 73 and 23% of control 24 hr, after BSO in subcutaneous and intracranial xenografts respectively. Concurrent measurement of total glutathione in the contralateral (non-tumor-containing) cerebral hemisphere in mice bearing intracranial D-54 xenografts demonstrated insignificant depletion of glutathione. Multiple doses of BSO, at 12-hr intervals, resulted in further depletion to 27% (s.c.) and 16.5% (i.c.) of control 12 hr following the final dose of BSO. Quantitative analysis of BSO delivery to xenograft and contralateral brain tissue revealed transfer constants, K1, of 15.8-24.1 x 10(-3) and 2.4 x 10(-3) ml.g-1.min-1 for xenograft and "normal" brain respectively. This highly selective depletion of glutathione in neoplastic tissue versus surrounding non-neoplastic host tissue may have therapeutic implications for the rational use of chemotherapeutic and radiotherapeutic intervention.

Animals↗
PubMed1988

Melphalan transport, glutathione levels, and glutathione-S-transferase activity in human medulloblastoma.

Melphalan transport, glutathione levels, and glutathione-S-transferase activity were measured in two continuous human medulloblastoma cell lines and transplantable xenografts in athymic nude mice, TE-671 and Daoy. In vitro mean glutathione levels were 10.06 nmol/10(6) cells in TE-671 and 2.96 nmol/10(6) cells in Daoy. In vitro mean glutathione-S-transferase values were 91.52 nmol/min/mg protein in TE-671 and 50.31 nmol/min/mg protein in Daoy. Transport studies revealed kinetic parameters of Km = 108.3 microM, Vmax = 363.1 pmol/10(6) cells/min in TE-671 and Km = 111.7 microM, Vmax = 180.6 pmol/10(6) cells/min in Daoy. Melphalan transport was inhibited by both DL-alpha-2-aminobicyclo[2.2.1]heptane-2- carboxylic acid and sodium ion depletion in TE-671 and Daoy cells in vitro, indicating that both systems of amino acid transport are functional in these medulloblastoma lines. In vivo s.c. xenograft glutathione values were lower (7.79 nmol/mg protein) in TE-671 than in Daoy (13.68 nmol/mg protein). The mean plasma concentration in mice given a 10% lethal dose (71.3 mg/m2) of melphalan i.p. was 50.3 microM at 10 min, with the half-life of 29.9 min. At this dose, s.c. xenograft levels were 2- to 3-fold higher in TE-671 than in Daoy tumors for the 3-h period measured. These studies demonstrate transport parameters confirming facilitated transport of melphalan in human medulloblastoma, a mean murine plasma melphalan concentration (following treatment with melphalan) above the in vitro drug dose at which there is a 90% reduction in the number of colonies in comparison to controls for TE-671 and Daoy for 2 h, and glutathione and glutathione-S-transferase levels in the same range previously reported in other melphalan-sensitive and melphalan-resistant human tumors. Future work with spontaneous and acquired melphalan-resistant human medulloblastoma cell lines and xenografts will define the role of these mechanisms in mediating drug resistance.

Animals↗
PubMed1988

Response of radiation-induced fibrosarcoma-1 in mice to cyclophosphamide monitored by in vivo 31P nuclear magnetic resonance spectroscopy.

In vivo 31P nuclear magnetic resonance spectroscopy has been used to examine the RIF-1 fibrosarcoma in mice during untreated growth and following chemotherapy with cyclophosphamide. Levels of inorganic phosphate increase relative to phosphocreatine or nucleoside triphosphates during early untreated growth. After the tumor reaches a volume of approximately 1 g, no further decrease in energy level is observed. Following treatment with cyclophosphamide, tumor phosphorus metabolite ratios and pH are significantly altered, compared to untreated age-matched controls. During the growth delay period following chemotherapy there is a significant reduction in the ratio of inorganic phosphate to other phosphate metabolites, compared to age-matched controls. In addition, a more alkaline pH is observed in the tumors of treated animals. When the growth delay period ends, nuclear magnetic resonance spectra return to pretreatment patterns. The magnitude of the differences in 31P nuclear magnetic resonance spectral parameters between treated animals and untreated controls is dose dependent. However, doses of cyclophosphamide above 200 mg/kg do not result in earlier spectroscopic alterations, nor in larger effects by Day 3 after treatment, even though clonogenic cell killing and growth delay are greater at these higher doses.

Animals↗
PubMed1988

Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells.

The cytosolic aldehyde dehydrogenase (ALDH) isozyme from cyclophosphamide (CPA) resistant L1210 cells (L1210/CPA) was purified to apparent homogeneity using ternary enzyme complex-dye ligand chromatography. The purified isozyme migrates as a single band at Mr 51,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis and as a single charge species at isoelectric point = 5.8 in isoelectric focusing. Micromolar Km values were estimated with both propionaldehyde (Km = 5 microM) and 4-hydroxy cyclophosphamide (4-OH CPA) (Km = 4 microM) as substrates, indicating that this isozyme is capable of oxidizing the activated cyclophosphamide intermediate 4-hydroxy CPA/aldophosphamide to carboxyphosphamide. This isozyme is also potently inhibited by disulfiram (Ki = 6 microM) and 4-(diethylamino)benzaldehyde (Ki = 0.04 microM). Both of these inhibitors are capable of sensitizing L1210/CPA cells to activated CPA in clonogenic survival assays. Thus, the increased levels of only the cytosolic ALDH isoform in L1210/CPA cells appear to be the single phenotypic difference necessary for conferring resistance to CPA. Monospecific antibodies to the L1210/CPA isozyme have been used in Western blot analysis to detect nanogram levels of ALDH in cell and tissue extracts. These antibodies cross-react with the cytosolic isozyme in P388/CPA cells, mouse liver, mouse small intestine, and the 1C1C7 hepatoma cell line, whereas no ALDH is detected in sensitive L1210 or P388 cells. Also, these antibodies show little cross-reactivity with the mitochondrial isozyme from mouse liver or 1C1C7 cells. From immunological and inhibitor characterization, the soluble ALDH isozyme in L1210/CPA cells appears identical to the normal mouse tissue isozyme.

Aldehyde Dehydrogenase↗
PubMed1988

Role for aldehyde dehydrogenase in survival of progenitors for murine blast cell colonies after treatment with 4-hydroperoxycyclophosphamide in vitro.

We have studied the effects of 4-hydroperoxycyclophosphamide (4-HC) on murine hemopoietic progenitors. We found dose-dependent killing and differential sensitivities of colony forming cells with burst forming units-erythrocyte being most sensitive and colony-forming units-granulocyte/erythrocyte/macrophage/megakaryocyte most resistant. We also tested the effects of 4-HC on more primitive murine progenitors which were identifiable in our assay system when the addition of interleukin-3 was delayed until Day 7. We found that the sensitivities of the progenitors for blast cell colonies are similar to those of colony-forming units-granulocyte/erythrocyte/macrophage/megakaryocyte and that late-appearing blast cell colonies were particularly resistant to 4-HC. In order to study the mechanism of differential sensitivities of murine progenitors to 4-HC, we examined the sensitivities of murine progenitors to 4-HC after brief incubation with diethylaminobenzaldehyde, an inhibitor of aldehyde dehydrogenase. Progenitors for granulocyte/macrophage colonies, granulocyte/erythrocyte/macrophage/megakaryocyte colonies, and blast cell colonies became more sensitive to 4-HC and the differential sensitivities of the progenitors disappeared following this treatment. We also tested the sensitivities of the progenitors to phenylketophosphamide, an analogue of 4-HC which is resistant to inactivation by aldehyde dehydrogenase. Various colony-forming units exhibited a similar dose response to this compound. These data indicate that intracellular levels of aldehyde dehydrogenase might play an important role in differential sensitivities of murine colony-forming units to 4-HC.

Aldehyde Dehydrogenase↗
PubMed1988

Enzymatic mechanisms of resistance to alkylating agents in tumor cells and normal tissues.

The presence in tumor cells and in normal cells of enzymes which metabolize and inactive alkylating agents appears to play a major role in determining the effectiveness of alkylating agents against human tumors and the toxicities of these agents to normal tissues. The enzyme aldehyde dehydrogenase appears to protect bone marrow and the gastrointestinal tract against toxicity from cyclophosphamide and other closely related oxazophosphorine agents. The presence of this enzyme in bone marrow stem cells facilitates the elimination of tumor cells from bone marrow suspensions, with preservation of the ability of the marrow suspension to reconstitute normal hematopoiesis in a patient. A variety of mouse and human tumors has been shown to be resistant to cyclophosphamide on the basis of an elevated aldehyde dehydrogenase content. The clinical significance of this type of resistance is currently being explored. Increased levels of glutathione-S-transferase have been shown to be associated with cellular resistance to a variety of alkylating agents. We have identified and characterized the conjugates of nitrogen mustards with glutathione. The formation of these conjugates is catalyzed by glutathione-S-transferase. The further study and characterization of these specific reactions should contribute to the understanding and quantitation of this type of alkylating agent resistance.

Aldehyde Dehydrogenase↗
PubMed1988

Labeled plasma metabolites of L-methyl-hydrogen-3-methionine and L-methyl-carbon-14-methionine in the dog.

The validity of the mathematical models that attempt to describe positron emission tomography (PET) images produced with [11CH3] methionine in terms of rates of local cerebral protein synthesis has yet to be established. A major objection to current models is that the use of methionine labeled at the methyl position results in the dispersal of the label among various methyl-accepting compounds that appear in the plasma and may then enter the brain. One approach to overcoming this problem has been the use of "standard" corrections for the activity contributed to plasma by labeled plasma protein and labeled serine. In order to determine the validity of this approach, the metabolic fate of labeled methionine was studied in six dogs. After injection with either [C3H3] methionine or [14CH3] methionine arterial blood was sampled. Plasma fractions containing protein were separated by fast gel filtration, counted with standard scintillation techniques, and their radioactivity was compared with total plasma radioactivity. Plasma was also separated by high-pressure liquid chromatography into methionine, serine, and nonmethionine or serine-containing fractions. These fractions were counted, and their radioactivity was compared with total plasma radioactivity. Labeled protein appeared in plasma about 20 minutes postinjection and then increased steadily. Labeled serine also appeared and reached a peak value of 9.4 +/- 2.1% of plasma activity at 40 minutes. Of greatest interest was the appearance in later plasma samples of increasing amounts of activity contained in nonserine low molecular weight metabolites of methionine. At 40 minutes, those metabolites made up 27 +/- 6.9% of total plasma activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Variability in 4-hydroperoxycyclophosphamide activity during clinical purging for autologous bone marrow transplantation.

We examined the effects of varying incubation conditions on the in vitro activity of 4-hydroperoxycyclophosphamide (4HC). 4HC activity against CFU-GM and against the K562 tumor cell line decreased with increasing the RBC concentration of the incubation mixture. Increasing the concentration of nucleated bone marrow cells in the incubation mixture also decreased the 4HC activity. Evaluation of 53 consecutive patients undergoing autologous bone marrow transplantation (BMT) revealed that the incubation RBC concentration during clinical purging showed a similar effect on CFU-GM recovery. Aldehyde dehydrogenase content of RBCs and nucleated marrow cells appears to be the cause of the inhibition of 4HC activity. Although there was no difference in individual CFU-GM sensitivity to 4HC among normals, previously treated patients undergoing autologous BMT showed significant variability in CFU-GM sensitivity to 4HC. The combined effects of incubation RBC concentration and individual patient 4HC sensitivity appear to account for most of the variability in CFU-GM recovery and speed of hematologic recovery after clinical purging with 4HC.

Bone Marrow↗