Free radicals in health and disease.
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Free Radicals↗
Biomedical subjects
A Petkau
Publications and source records attributed to A Petkau.
Protection of bone marrow progenitor cells by superoxide dismutase.
Intravenously administered cuprozinc-superoxide dismutase in X-irradiated mice hastens the recovery of peripheral blood cells. This effect is consistent with protection of the pluripotent stem cells by the enzyme. Amongst the bone marrow cells committed to differentiation along the myeloid pathway, there exists in mice a subpopulation of macrophage progenitor cells that is inactivated by superoxide radicals, generated photochemically or by X-rays. This cell killing effect is inhibited by superoxide dismutase, in part because it acts intracellularly. Human bone marrow also contain a superoxide-sensitive subpopulation of myeloid progenitor cells that is protected by superoxide dismutase but not by catalase. As well, human myeloid progenitor cells contain a subpopulation with enhanced sensitivity to X-rays in vitro. Treatment of these cells with exogenous superoxide dismutase reduces the sensitivity to X-rays by a factor of 2.
Animals↗
Enrichment of progenitor cells from human bone marrow by flow cytometry.
Mononuclear cells, harvested from fresh human bone marrow specimens by density gradient separation, were suspended in phosphate buffered saline and analyzed by flow cytometry in terms of the forward and right angle scattering of the incident light. The rectilinear distribution, obtained by plotting the intensity of light scattered in the forward and right angle directions, contained three regions of interest in which the percentage of cells (Mean +/- standard deviation) with respect to the total was as follows: Region 1: 17.6 +/- 9.9; region 2: 5.3 +/- 1.4; region 3: 71.7 +/- 9.4. Cells from each region were sorted by flow cytometry and plated in semi-solid agar containing cell conditioned medium supportive of myeloid colony formation. Cells from region 2 contained the majority of progenitor cells that gave rise to such colonies at a plating efficiency that rose in proportion to the extent by which the region 2 cells in samples was increased through sorting. This increase in plating efficiency was 6 to 43 fold. Thus, region 2 of the cytometric distribution of cells from normal, unstained human bone marrows was a good source of myeloid progenitor cells.
Bone Marrow Cells↗
Response of human bone marrow progenitor cells to X-rays in vitro.
In 25 out of 33 cases the survival response of myeloid progenitor cells from fresh human bone marrows, X-irradiated in vitro in phosphate-buffered saline at 23 degrees C, was characterized by a two-term exponential relationship, with D0 values of 0.42 +/- 0.19 Gy and 1.38 +/- 0.37 Gy, respectively. In the remaining eight cases for which the colony-forming efficiency was 10 times lower, survival followed a single exponential function with D0 = 1.18 +/- 0.15 Gy. The biphasic response at 23 degrees C became a single exponential response when the temperature at irradiation was 30 or 37 degrees C.
Bone Marrow↗
A calorimetric study of human CuZn superoxide dismutase.
Structural alterations, as manifested by thermal transitions, caused by removal or binding of metal ions to human and bovine CuZn superoxide dismutases (SODs) were investigated by differential scanning calorimetry. Although holo forms of the two mammalian enzymes exhibited irreversible thermal transitions (delta Hcal. = 27.7 J/g and Td = 104 degrees C for bovine SOD; delta Hcal. = 23.6 J/g and Td = 101 degrees C for human SOD), only the bovine apoenzyme showed the presence of a less thermostable form (delta Hcal. = 10.7 J/g and Td = 63 degrees C). These observations suggested that human apo-SOD had considerably less conformational order than bovine apo-SOD. Reconstitution of human and bovine apoenzymes with Cu2+ and Zn2+ resulted in recovery of thermodynamic parameters and specific activity. Binding of Zn2+ alone to human apo-SOD resulted in the formation of two distinct structural units, detectable by differential scanning calorimetry, which underwent conformational disorder at 82 and 101 degrees C respectively. Saturation of binding sites with both Zn2+ and Cu2+ appeared to stabilize the enzyme structure further as shown by elimination of the low-temperature transition and the appearance of another thermal transition at a higher temperature.
Apoenzymes↗
In vitro response of human bone marrow progenitor cells to superoxide radicals.
Human bone marrow progenitor cells, grown on solid agar, developed colonies of the white blood cell series, principally granulocytes, monocytes and macrophages. Following exposure to superoxide radicals, generated photochemically, a fraction of the progenitor cells was inactivated as evidenced by loss of colony formation. The loss in proliferative capacity depended on the illumination time and individual nature of the donated specimens. Protection of the progenitor cells by active superoxide dismutase depended on the illumination time and temperature. Inactive superoxide dismutase and active catalase did not protect the cells from the photochemical inactivation process.
Colony-Forming Units Assay↗
Role of superoxide dismutase in modification of radiation injury.
The effects of superoxide dismutase on radiobiological end-points are discussed in terms of the time scale of primary radiation chemical reactions and later cellular and physiological processes. The effectiveness of SOD on responsive subpopulations of bone marrow progenitor cells is shown to be temperature- and dose-dependent. Under certain conditions, the protective actions of superoxide dismutase and catalase complement each other in a sequential fashion. Finally, cellular levels of endogenous superoxide dismutase in atomic radiation workers are compared with those in a control population.
Animals↗
Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography.
Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.
Chelating Agents↗
Scientific basis for the clinical use of superoxide dismutase.
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Animals↗
Radioprotection by superoxide dismutase of macrophage progenitor cells from mouse bone marrow.
X-ray survival of cultured macrophage progenitor cells from mouse bone marrow was represented by a two-component curve, both in the absence and presence of superoxide dismutase. Protection by the enzyme was limited to the radiosensitive fraction, for which a dose modifying factor of 2.8 +/- 0.7 was obtained. Catalase did not protect. Survival of the radiosensitive fraction, with and without exogenous superoxide dismutase, was temperature-dependent, whereas that of the radioresistant fraction was not. In the former case, the energy required for the enzyme-treated cells was approximately 13kJ/mol.
Animals↗
Simulated responses to intermittent lognormally distributed doses at variable dose rates.
Two previously reported equations for low-LET radiation, R = DmI-n and R = D[a + b(lnI) + h(lnI)2], where the response (R) is expressed in terms of dose (D) and dose rate (I) and the other parameters are constants, were used to compute distributions of response to intermittent, low-level, lognormally distributed doses, administered at different dose rates. The simulations show that the means of the response distributions are affected by both the dose and dose rate distributions. Within certain ranges of their respective values, the influence of the dose and dose rate on the response are in opposite directions, and lead to similar response distributions for numerically different combinations of dose and dose rate. The resolution and mean values of response distributions, when based on nonlinear dose-response relationships, are also affected by dose fractionation.
Animals↗
Small-angle neutron scattering from native and irradiated superoxide dismutase in aqueous solution.
The approximate size and shape of holo and apo forms of bovine cupro-zinc erythrocyte superoxide dismutase (EC 1.15.1.1) were determined by small-angle neutron scattering from aqueous solutions at neutral pH. A model assuming a cylindrical shape gave the best fit to the data for both forms of the enzyme. The radius of gyration, Rg, of the apoenzyme was found to be marginally larger than that of the holoenzyme. Scattering from the protein vanished for H2O/2H2O mixtures containing 42(+/- 2)% 2H2O, and the negligible dependence of Rg on the 2H2O fraction indicated uniform scattering density. Irradiation with 60Co gamma-rays resulted in aggregation of superoxide dismutase molecules; scattering at small doses was interpreted in terms of pairwise side-by-side aggregation. For large doses (approx. 3.8 X 10(3) Gy) and at relatively high enzyme concentrations (320 microM), the interpretation of the neutron scattering data is ambiguous. The value of Rg suggests that end-to-end stacking of the cylindrical molecules is one possibility. The equilibrium concentration of separated subunits was found to be unobservable and calculations showed that denaturation did not contribute significantly to our neutron scattering measurements for the radiation doses employed (maximum 3.8 X 10(5) Gy). High-performance liquid chromatography (HPLC) data showed that in mixtures the apo and holo forms of superoxide dismutase interact with one another, and that the side-by-side aggregates, induced by irradiation of the enzyme, are readily dissociated, resulting in a single elution peak that is resolved from the later peak common to both the apo and holo forms.
Animals↗
Identifying superoxide-sensitive progenitor cells in mouse bone marrow.
Macrophage progenitor cells in bone marrow, that develop into attached colonies in liquid culture medium, contain a fraction of cells sensitive to photochemically generated superoxide radicals. This fraction varies from one animal to another. Populations of cells containing the superoxide-sensitive fraction show a greater sensitivity to X-rays than do populations in which this fraction has been photochemically inactivated. The change in radiosensitivity was proportional to the superoxide-sensitive fraction.
Animals↗
Simulated responses to lognormally distributed continuous low radiation doses.
Three previously reported equations, expressing response as a function of both dose and dose rate, for low LET radiation were used to compute distributions of response to continuous, low-level, lognormally distributed doses for populations of variable size up to 5000. The simulations show that, when the response is inversely dependent on dose rate, the mean of the response distribution can either increase or decrease with increasing exposure to radiation, depending on whether or not the dose-dependent term is the dominant one in an equation. In one equation, which defines the response as conjugated hydroperoxide formation in irradiated membranes, the magnitude of the dose-dependent term is reduced the most by superoxide dismutase, an enzymic inhibitor of free radical processes. This results in the means of response distributions decreasing with increasing dose from continuous, low-level radiation.
Animals↗
Extension of kinetic model for superoxide dismutase inactivation to the irradiated manganese tetramer.
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Cobalt Radioisotopes↗
Radiation-induced inactivation of superoxide dismutase in nitrous oxide-saturated solutions. A kinetic model.
It has been reported that the inactivation yield of superoxide dismutase from bovine erythrocytes irradiated in N2O-saturated solutions increases exponentially with the initial enzyme concentration and that dose-response curves are non-exponential. The present work analyses these data using a kinetic model which indicates that the inactivation process is a second order reaction and that the reactivities of the active (Ea) and inactive (Ei) enzymes toward hydroxyl radical (.OH) are unequal. The calculated rate constants for the reactivities of Ea and Ei with .OH are 2.54 . 10(10) M-1 . s-1, respectively. The ratio between number of collisions and inactivation events is estimated at 20, in good agreement with a previous report. This model suggests that subunit exchange processes play an important role in the inactivation reaction.
Animals↗
Active oxygen and medicine. Introduction: Free radical involvement in physiological and biochemical processes.
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Animals↗
On the safety of treatment with bovine superoxide dismutase: production of a humoral antibody response in rabbits with repeated treatment.
Treatment of rabbits with sterile solutions of bovine Cu-Zn superoxide dismutase according to schedules recommended for radiation cystitis and radiation side effects has elicited the production of antibodies to the enzyme, detected in rabbit sera by a sensitive and specific radioimmunoassay. No antibodies were detected after a single treatment. However, on repeated administration of the enzyme all animals, including those that received 4 Gy whole body radiation prior to treatment, produced detectable antibody. Titers of antibody in sera rose progressively with repeated injections of the enzyme. Irradiation served merely to delay the onset of the immune response. In addition, human and bovine Cu-Zn superoxide dismutase have been found to be antigenically distinct when examined by competitive inhibition radioimmunoassay. These antigenic differences and the immunogenicity of the bovine enzyme in rabbits raise reasonable doubt as to the safety of administration of bovine superoxide dismutase to human patients. It is recommended that such patients be monitored for antibody production, preferably by radioimmunoassay.
Animals↗