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B D Michael

Publications and source records attributed to B D Michael.

At least 73 records · Page 4Linked to original sources
PubMed1991

The effects of counter-ion condensation and co-ion depletion upon the rates of chemical repair of poly(U) radicals by thiols.

Bimolecular rate constants for reactions of poly(U) radicals with a series of thiols of varying net charge (Z) were measured by pulse radiolysis with conductivity detection at low ionic strength. At pH 7 and 18 degrees C the values of k2 (M-1s-1) were: reduced glutathione (Z = -1), less than 500; 2-mercaptoethanesulphonic acid (Z = -1), 1.5 x 10(3); 2-mercaptoethanol (Z = 0), 1.8 x 10(5); cysteine (Z = 0), 2.0 x 10(5); cysteamine (Z = +1), 4.1 x 10(7). Values determined at pH 4 were: 2-mercaptoethanol, 6.1 x 10(5); cysteamine 2.2 x 10(8); N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, Z = +2), 4.6 x 10(8). The variation in rate with structure could not reasonably be attributed to inherent reactivity differences in the thiols and was ascribed to inhomogeneous distributions of the thiols in solution resulting from electrostatic interactions. Thus, cationic thiols are concentrated approximately 100-fold near poly(U), relative to neutral thiols, as a consequence of counter-ion condensation, whereas anionic thiols have approximately 100-fold lower concentration near poly(U) than neutral thiols as a result of co-ion depletion. These results show that the ability of a thiol to repair radical sites in a polyanion is dramatically influenced by its net charge as a consequence of the counter-ion condensation and co-ion depletion phenomena.

Cysteamine↗
PubMed1991

Rates for repair of pBR 322 DNA radicals by thiols as measured by the gas explosion technique: evidence that counter-ion condensation and co-ion depletion are significant at physiological ionic strength.

Rates of repair of pBR 322 plasmid DNA radicals by thiols of varying net charge (Z) at pH 7 and physiological ionic strength were measured using the oxygen explosion technique. The extent of conversion of supercoiled to relaxed circular plasmid was measured by HPLC as a function of the time of oxygen exposure before or after irradiation, the time-courses being fitted by a pseudo-first-order kinetic expression with k1 = k2[RSH]. Values of k2 (M-1 S-1) were: 2.1 x 10(5) (GSH, Z = -1), 1.4 x 10(6) (2-mercaptoethanol, Z = 0), 1.2 x 10(7) (cysteamine, Z = +1), 6.6 x 10(7) (WR-1065 or N-(2-mercaptoethyl)-1,3-diaminopropane, Z = +2). The approximately 6-fold increase in rate with each unit increase in Z is attributed to concentration of cationic thiols near DNA as a consequence of counter-ion condensation and reduced levels of anionic thiols near DNA owing to co-ion depletion. The results are quantitatively consistent with chemical repair as a significant mechanism for radioprotection of cells by neutral and cationic thiols under aerobic conditions, but indicate that repair by GSH will compete effectively with oxygen only at low oxygen tension.

Chromatography, High Pressure Liquid↗
PubMed1991

The oxygen effect: variation of the K-value and lifetimes of O2-dependent damage in some glutathione-deficient mutants of Escherichia coli.

Radiosensitization by various concentrations of O2 has been studied in an Escherichia coli K-12 wild-type strain and some derived glutathione (GSH)-deficient mutants using 60Co gamma-irradiation. The maximum oxygen enhancement ratio (OER) and the K-value, the O2 concentration that produced half the maximum O2 effect, were found to depend on the GSH biosynthetic capacity of the strains. For the GSH+ wild-type strain, AB1157, and the GSH- mutant, 830, which is deficient in glutathione synthetase, the final enzyme in the GSH biosynthetic pathway, the maximum OERs were both about 3.9 and the K-values were 0.53% and 0.24% O2, respectively. On the other hand, the maximum OERs for two GSH- mutants, 7 and 821, both deficient in gamma-glutamylcysteine synthetase, the penultimate enzyme in the GSH biosynthetic pathway, were about 2.7 and the K-values were about 0.06% O2 for both. The fast chemical repair of O2-dependent damage in these strains was measured using a fast mixing and irradiation method, the gas explosion technique. The chemical repair rates in the various E. coli strains varied approximately in proportion to the O2 K-values, and both the rates of chemical repair and the K-values correlated approximately with the levels of non-protein sulphydryls in the various strains.

Cobalt Radioisotopes↗
PubMed1990

The influence of experimental design and data analysis on the determination of recovery kinetics of radiation damage between acute dose-rate treatments in vivo.

Current interest in determining the rate of recovery of damage between radiation doses in fractionated treatments has resulted in the development of several experimental designs and methods of analysis to address this. One approach is where two or more fractions are given with a varying interval. Isoeffect doses are then determined from the dose-response curves for each interval, and these are plotted on a logarithmic axis against time on a linear scale. An estimate of the rate of dose recovery can then be made if the data show monoexponential or well-defined multiexponential kinetics. However, three problems can be identified in this simple protocol. First, most repair models (e.g. Thames' IR and Curtis' LPL) assume that between two doses loge (cell survival), i.e. underlying effect, not dose itself, recovers exponentially with time. Experimental data support this assumption. Since underlying effect and dose are not linearly related, recovery measured from the change in isoeffect dose can appear substantially slower (depending on dose per fraction) than the true underlying recovery rate of damage. This artifact is avoided by converting dose increments into changes in underlying effect (with the linear-quadratic model) or by measuring underlying effect more directly in 'top-up' experiments. The use of (neutron) top-up experiments is preferred, as it enables recovery between constant X-ray doses per fraction to be studied, and makes no prior assumptions regarding either the shape of the X-ray dose-response curve or how recovery takes place, although the shape of the neutron dose-response curve must be known. Second, plotting log (unrecovered damage) against time can overestimate recovery half-times, because such plots cannot handle negative values and therefore become naturally weighted in favour of the data from the longer time intervals where the difference from complete recovery is smallest. This problem is managed by using nonlinear regression to fit the values of unrecovered damage expressed on a linear scale against interval. Third, experiments using three or more evenly spaced fractions, 'concertina'-style, permit interaction between non-adjacent fractions. If this is not taken account of, then recovery appears to be initially faster and multiexponential, even though the underlying recovery may be actually monoexponential. Thus concertina experiments are poor at resolving the precise shape of recovery-kinetics profiles and are less suited for measuring any dependence of recovery rate on dose per fraction compared with approaches using either just two fractions, or two fractions per day.

DNA Repair↗
PubMed1990

The irradiation of V79 mammalian cells by protons with energies below 2 MeV. Part II. Measurement of oxygen enhancement ratios and DNA damage.

The effectiveness of low-energy (below 2 MeV) protons at inducing DNA damage in the form of single- and double-strand breaks has been determined. Protons with mean energies of 1.90 MeV, 1.15 MeV and 0.76 MeV corresponding to track average LETs of 17 keV/microns, 24 keV/microns and 32 keV/microns, respectively, have been used and compared with 250 kVp X-rays and 3.8 MeV 238Pu alpha-particles. Although there was variation in the RBE for DNA ssb induction with LET, the RBEs for dsb induction at all three proton energies and for 3.8 MeV alpha-particles were all around 1.0. This suggests that, if DNA dsb are important in radiation-induced cell lethality, the probability of an induced dsb leading to a lethal event increases with increasing LET of radiation. Oxygen enhancement ratios were measured for both cell survival and DNA dsb induction, and in both cases a decrease in OER with increasing LET was observed.

Animals↗
PubMed1990

Fast kinetics of the oxygen effect for DNA double-strand breakage and cell killing in irradiated yeast.

Lifetimes of oxygen-dependent precursors of DNA double-strand breaks (dsb), as determined by applying the gas explosion technique, were found to be dose-dependent. The analysis of data for dsb induction obtained in diploid 211*B cells by the neutral sucrose sedimentation technique exhibits a half-life of 2.18 ms at a pulse dose of 500 Gy. Lifetimes are also obtained indirectly by analysing the inactivation of diploid rad54-3 cells, which are defective in the rejoining of dsb when incubated at 36 degrees C. The half-life at a pulse dose of 40 Gy is only 0.25 ms. Since the cell size of both strains is very similar, the nine-fold longer lifetime determined in cells of strain 211*B may be caused by radiolytic depletion of glutathione due to the 12-fold higher dose applied to these cells. Therefore, the lifetime measured by the inactivation of rad54-3 cells (36 degrees C) is considered to be more relevant than that obtained by direct measurements of dsb. The influence of dsb rejoining on the fast kinetics of the oxygen effect was studied using rad54-3 cells, which are capable of rejoining dsb when incubated at 23 degrees C. When allowance for dsb rejoining was made, two components become detectable with half-lives of 0.75 and 29 ms. Haploid yeast cells in stationary phase are not capable of rejoining dsb. Using such cells proficient or deficient in the synthesis of glutathione (GSH), the lifetimes were found to be 0.37 or 0.49 ms for GSH-proficient (100 per cent GSH) and -deficient (2.1 per cent GSH) cells, which is in agreement with the view that chemical restitution of oxygen-dependent precursors is impaired in gsh- cells.

Cells, Cultured↗
PubMed1989

Cell killing and DNA damage in Chinese hamster V79 cells treated with hydrogen peroxide.

Hydrogen peroxide induces lesions in cells similar to those from ionizing radiation, by a Fenton-type production of hydroxyl radicals. At 4 degrees C significant levels of DAN single-strand breaks (ssb) could be measured using the alkaline elution technique, after a 20-min incubation with 10(-5) mol dm-3 H2O2. Only at higher concentrations of H2O2 (greater than 10(-4) mol dm-3) where the levels of ssb measured corresponded to that induced by more than 18 Gy of X-rays, was any significant cell killing detected in a clonogenic assay. Cell killing was observed to coincide with the measurement of significant levels of DNA double-strand breaks (dsb) using the filter elution technique at pH 9.6. This suggests that dsb and not ssb are important as regards hydroxyl-radical-induced cell kill, as found for ionizing radiation. The correlation of induced dsb with lethal events showed that the predicted lethal effect of the H2O2-induced dsb was approximately 5 times less than that of X-ray-induced dsb. This is compared with data previously obtained which showed differences in the lethality of dsb with the quality of radiation (Prise et al. 1987).

Animals↗
PubMed1989

A single-shot rapid-mixing device for radiobiological studies with mammalian cells.

A single-shot rapid-mixing device is described for the rapid addition of solutions of radiation-modifying agents, to cell suspensions, at well-defined times relative to a pulse of radiation. The liquid injection system could be used to initiate or quench a wide range of chemical or biochemical reactions. The rapid-mixing device is based on a syringe driven by a stepper motor and can inject up to 2 cm3 liquid in less than 100 ms. The radiation source, a 4 MV Van de Graaff accelerator, provides an electron beam which is deflected from the beam dump on to the sample in two stages, providing a 10 ms radiation pulse. A digital delay circuit defines the interval between mixing and irradiation. The apparatus has been designed to study the kinetics of processes that occur over a time range extending from about 0.1 s to some minutes. It bridges the gap between the ranges available with conventional fast-mixing and those using standard X- or gamma-irradiation methods. The time resolution of the technique has been examined by following the timecourse of radiosensitization by oxygen in mammalian cells. The timecourse of radioprotection of aerobic mammalian cells by dithiothreitol has been measured using the technique.

Animals↗
PubMed1989

The irradiation of V79 mammalian cells by protons with energies below 2 MeV. Part I: Experimental arrangement and measurements of cell survival.

The relative biological effectiveness (RBE) has been determined for protons with mean energies of 1.9, 1.15 and 0.76 MeV, from measurements of the survival of V79 Chinese hamster cells. The cells are supported as a monolayer and are swept through a beam of scattered protons produced using a 4 MeV Van de Graaff accelerator. An estimation of the dose and unrestricted linear energy transfer (LET) variation within the sensitive volume of the cells is given for the three proton energies. The RBEs for cell survival (relative to 250 kVp X-rays) at the 10 per cent survival level are 1.6, 1.9 and 3.36 for protons with track-average LETs of 17, 24 and 32 keV microns-1 respectively, and the data suggest that protons are most effective at about 40-50 keV microns-1. It is shown that the proton RBEs can be reconciled with those of other light ions if plotted against z*2/beta 2 (where z* is the effective charge and beta is the relative velocity) rather than against LET.

Animals↗
PubMed1989

Non-linear dose-effect curve for DNA double-strand breaks by low LET radiation: the effect of eluting buffer composition on the measurement of breaks by the filter elution technique.

Chinese hamster V79 cells were irradiated with 250 kVp X-rays and analysed for the presence of DNA double-strand breaks using filter elution. Changing the pH of the elution and lysis buffers from pH 9.6 to 7.2 leads to a decreased rate of elution of the DNA from the filters and a small difference in the dose-effect relationship obtained. In both cases the dose-effect curves were non-linear. When cells are irradiated with 238Pu alpha-particles a difference in the rate of elution of the DNA is also observed between the two pH values, although the dose-effect curves are linear. These findings suggest that there may be qualitative differences in the types of lesions being induced by low-LET radiation and being scored as dsb in the filter elution assay at pH 9.6. However, carrying out elution at pH 7.2 does not eliminate the nonlinear response measured after X-irradiation.

Alpha Particles↗
PubMed1989

Measurement and analysis of supralinearity in LiF TLD-100 irradiated by 1.5 keV x-rays.

Measurements of the response of lithium fluoride TLD-100 to 1.5 keV ultrasoft x-rays, 240 keV x-rays and 137Cs gamma-rays at doses below 70 Gy are presented and the implications for the track interaction model are discussed. This model proposes that thermoluminescence is due to the combination electrons and holes formed along secondary charged particle tracks. As the dose is increased, the average distance between particle tracks is reduced and there is a greater probability of combination between neighbouring tracks; this mechanism is used to explain the supralinear response of lithium fluoride to certain radiations. Measurements using ultrasoft x-rays are interesting in this context because the energy from each photon is deposited in a small volume, therefore the probability of combination between neighbouring events should only be significant at high doses. The results, however, show that lithium fluoride exhibits a significant supralinear response to 1.5 keV x-rays after only a few tens of grays and suggests that the track interaction model alone cannot explain the phenomenon of supralinearity.

Cesium Radioisotopes↗
PubMed1989

Measurements of neutron energy using a recoil-proton telescope and a high-pressure ionization chamber.

Two very different techniques for measuring the energy of neutrons in the energy range 0.1-10 MeV are presented and compared. A recoil-proton spectrometer is used to determine the energy spectra of neutrons produced by the d(4)-Be and p(4)-Be reactions down to the low-energy threshold of 0.7 MeV. The same radiation fields are also measured with a recently developed method using a high-pressure ionization chamber that can be used to determine the mean energy of the neutrons in a mixed neutron-gamma radiation field provided the gamma-ray absorbed dose fraction is determined independently. An intercomparison of the two methods shows that the high-pressure ionization chamber compares well and supplements the established recoil-proton spectrometer technique. The almost isotropic response of the chamber has enabled measurements to be made of the variation of mean neutron energy with depth in water for the two radiation fields.

Fast Neutrons↗
PubMed1989

Measurement of DNA damage and cell killing in Chinese hamster V79 cells irradiated with aluminum characteristic ultrasoft X rays.

Chinese hamster V79 cells were irradiated with 1.487 keV aluminum characteristic X rays produced using a cold-cathode discharge tube. Under aerobic conditions a relative biological effectiveness (RBE) of 2.18 for cell killing in comparison to 250-kVp X rays was measured using cells grown in suspension and irradiated on membrane filters. DNA damage in the form of single-strand (ssb) and double-strand breaks (dsb) was measured using the filter elution technique. The aerobic RBEs are 1.64 for dsb induction and 0.49 for ssb induction, consistent with the view that dsb are more closely related to cell kill than ssb. A reduced oxygen enhancement ratio (OER) for cell killing was measured for Al-K X rays, but the OER for dsb induction was similar to that measured for 250-kVp X rays. A curvilinear relationship between dsb induction and dose is observed, similar to that seen for 250-kVp X rays. This agrees with the concept that ultrasoft X rays produce critical lesions similar to hard X rays but with a greater efficiency per unit dose.

Animals↗
PubMed1987

The relationship between radiation-induced DNA double-strand breaks and cell kill in hamster V79 fibroblasts irradiated with 250 kVp X-rays, 2.3 MeV neutrons or 238Pu alpha-particles.

Using the neutral filter elution technique, the induction of DNA double-strand breaks (dsb) has been measured in 250 kVp X-irradiated V79-379A Chinese hamster cells irradiated under air or nitrogen. The dose-effect curves for induced dsb were curvilinear, mirroring cell survival curves, such that there was an approximately linear relationship between induced dsb and lethal lesions (-In (cell survival)) which was independent of oxygen. With cells irradiated with 2.3 MeV neutrons or 238Pu alpha-particles the correlations between lethal events and dsb, although also approximately linear, do not match those for X-rays. With neutrons there is approximately a 2.5-fold reduction in the level of dsb induction per lethal event. Thus either the apparently linear relationships found are spurious, and there is no general correlation between induced dsb and lethal effect, or there are qualitative differences between neutron, alpha-particle and X-ray induced dsb that give them differing probabilities of cell kill.

Alpha Particles↗
PubMed1987

An arrangement for irradiating cultured mammalian cells with aluminium characteristic ultrasoft x-rays.

Ultrasoft x-rays are useful for testing the validity of mechanistic models of biological damage caused by radiation. Described here is the construction and operation of a cold-cathode transmission-target discharge tube for irradiating mammalian cells in vitro with aluminium characteristic x-rays (1.487 keV). Particular attention is given to the problems of sample preparation and dosimetry for this shallowly penetrating radiation. The proportion of contaminating bremsstrahlung radiation is measured to establish the optimum operating conditions. Preliminary data from experiments using V79 Chinese hamster cells show that aluminium characteristic x-rays are about twice as effective at inactivating the cells as 250 kVp x-rays.

Aluminum↗