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B D Michael

Publications and source records attributed to B D Michael.

At least 55 records · Page 3Linked to original sources
PubMed1997

Genomic instability in Chinese hamster cells after exposure to X rays or alpha particles of different mean linear energy transfer.

Evidence has accumulated that radiation induces a transmissible persistent destabilization of the genome, which may result in effects arising in the progeny of irradiated but surviving cells. An enhanced death rate among the progeny of cells surviving irradiation persists for many generations in the form of a reduced plating efficiency. Such delayed reproductive death is correlated with an increased occurrence of micronuclei. Since it has been suggested that radiation-induced chromosomal instability might depend on the radiation quality, we investigated the effects of alpha particles of different LET by looking at the frequency of delayed micronuclei in Chinese hamster V79 cells after cytochalasin-induced block of cell division. A dose-dependent increase in the frequency of micronuclei was found in cells assayed 1 week postirradiation or later. Also, there was a persistent increase in the frequency of dicentrics in surviving irradiated cells. Moreover, we found an increased micronucleus frequency in all of the 30 clones isolated from individual cells which had been irradiated with doses equivalent to either one, two or three alpha-particle traversals per cell nucleus. We conclude that the target for genomic instability in Chinese hamster cells must be larger than the cell nucleus.

Alpha Particles↗
PubMed1996

Inactivation of V79 cells by low-energy protons, deuterons and helium-3 ions.

Previous work by ourselves and by others has demonstrated that protons with a linear energy transfer (LET) about 30 keVmum(-1)are more effective at killing cells than doubly charged particles of the same LET. In this work we show that by using deuterons, which have about twice the range of protons with the same LET, it is possible to extend measurements of the RBE of singly charged particles to higher LET (up to 50 keVmum(-1). We report the design and use of a new arrangement for irradiating V79 mammalian cells. Cell survival measurements have been made using protons in the energy range 1.0-3.7 MeV, deuterons in the energy range 0.9-3.4MeV and 3He2+ ions in the energy range 3.4-6.9 MeV. This corresponds to volume-averaged LET (within the cell nucleus) between 10 and 28 keVmum(-1) for protons, 18-50 keVmum(-1) for deuterons, and 59-106 keVmum(-1) for helium ions. Our results show no difference in the effectiveness of protons and deuterons matched for LET. However, for LET above about 30 keVmum(-1) singly charged particles are more effective at inactivating cells than doubly-charged particles of the same LET and that this difference can be understood in terms of the radial dose distribution around the primary ion track.

Animals↗
PubMed1996

A multiple-radical model for radiation action on DNA and the dependence of OER on LET.

We have developed a multiple-radical model of the chemical modification reactions involving oxygen and thiols relevant to the interactions of ionizing radiations with DNA. The treatment is based on the Alper and Howard-Flanders equation but considers the case where more than one radical may be involved in the production of lesions in DNA. This model makes several predictions regarding the induction of double strand breaks in DNA by ionizing radiation and the role of sensitizers such as oxygen and protectors such as thiols which act at the chemical phase of radiation action via the involvement of free radicals. The model predicts a decreasing OER with increasing LET on the basis that as radical multiplicity increases so will the probability that, even under hypoxia, damage will be fixed and lead to lesion production. The model can be considered to provide an alternative hypothesis to those of "interacting radicals' or of "oxygen-in-the-track'.

Animals↗
PubMed1995

Role of charge in the radioprotection of E. coli by thiols.

The role of net charge (zeta) of thiols in their ability to radioprotect cells has been investigated in a glutathione (GSH)-deficient strain of E. coli. This strain, 7, is deficient in the enzyme gamma-glutamylcysteine synthetase and allows the effects of added low molecular weight thiols to be studied. Using the gas explosion system it is possible to measure the chemical repair of the free-radical precursors of lethal lesions by thiols in intact cells. The first-order chemical repair rate in strain 7 is 280 s-1 in comparison with 1100 s-1 in the wild-type strain 1157. From the measured difference in the intracellular concentration of GSH between the wild-type and the mutant, this gives a second-order repair rate, kr, of 1.23 +/- 0.3 x 10(5) dm3mol-1s-1. Measurement of intracellular thiol levels after addition of various low molecular weight thiols showed that uptake was rapid, leading to stable thiol levels within 1 min. The ratios of the intracellular to extracellular concentrations (Cin/Cout) were 0.74 for 3-mercaptopropionic acid (zeta = -1), 0.56 for 2-mercaptoethanol (zeta = 0), 1.47 for cysteamine (zeta = +1) and 1.04 for WR1065 (zeta = +2). The kr's for these thiols were 1.3 +/- 0.5 x 10(5) dm3mol-1s-1 for 30-mercaptopropionic acid, 3.3 +/- 1.6 x 10(5) dm3mol-1s-1 for 2-mercaptoethanol, 3.9 +/- 1.1 x 10(5) dm3mol-1s-1 for cysteamine and 2.7 +/- 1.1 x 10(6) dm3mol-1s-1 for WR1065. These are lower and increase less with charge than previously published values for chemical repair in isolated pBR322 DNA, probably because of the association of nucleoproteins and polyamines with the cellular DNA of E. coli. However, the approximate three-fold increase in kr per unit increase in zeta shows that the counter-ion condensation and co-ion depletion are important in determining the effectiveness of charged thiols in the radioprotection of E. coli.

DNA Repair↗
PubMed1994

Effect of radiation quality on lesion complexity in cellular DNA.

Understanding the critical lesions induced by ionizing radiation in DNA and their relationship to cellular effects is an important challenge in radiation biology. Much evidence has suggested that DNA double-strand breaks (dsb) are important lesions. Establishing a cause and effect relationship between initial levels of DNA dsb, their repair rate or the level of residual unrepaired breaks, and cellular effects has proved difficult in mammalian cells. Several studies have measured yields of DNA dsb after irradiation with radiations of differing linear energy transfer (LET). In general the RBEs for dsb induction (20-100 keV/microns) have been lower than the RBEs measured for cell survival and in many cases are around 1.0. Several studies have shown differences in the rejoining of dsb with less dsb rejoined after high-LET irradiation in comparison with low-LET radiation. These results suggest that there may be differences in the types of lesions induced by different radiations and scored as DNA dsb using current techniques. Track structure modelling studies have suggested that some lesions induced will be clustered at the sites of energy depositions and that uniquely large energy deposition events are produced by high-LET radiations. Assays need to be developed to measure complex lesions in both model DNA and cellular systems. Different levels of complexity need to be considered such as clustering of radicals close to DNA, localized areas of DNA damage (1-20 bp) and lesions which may be induced over larger distances. Studies using new and existing assays of DNA damage, coupled with irradiation at various LETs, are directed at understanding the role of lesion complexity in relation to cellular effects.

DNA↗
PubMed1994

Action spectra for single- and double-strand break induction in plasmid DNA: studies using synchrotron radiation.

Ionizing radiations deposit a wide range of energies in and around DNA and this leads to a corresponding spectrum of complexity of the lesions induced. The relationships between the amount of energy deposited and the yields and types of damage induced are important in modelling the physical and chemical stages of radiation effect and linking them to biological outcome. To study these relationships experimentally, plasmids were mounted as a monolayer and exposed in vacuum to near-monoenergetic photons from the Daresbury Synchrotron. After irradiation, the DNA was washed off and assayed for single-(ssb) and double-strand breaks (dsb) using agarose gel electrophoresis. Dose-effect relationships for ssb and dsb induction were obtained at various energies in the range 8-25 eV. The initial responses in the low-dose region allowed damage yields to be estimated. However, a common feature is that the responses showed energy-dependent plateaus at higher doses as if a fraction of the DNA were shielded. Various measures were taken both to minimize and to correct for this effect. The data appear to show that the yields of ssb and dsb increase only slowly with photon energies > 10 eV, with a suggestion of similar threshold energies for both lesions. In the energy range covered, the yield of ssb is 12-20-fold greater than that of dsb. The data indicate that ssb and dsb may have a common precursor in this system. Earlier work with low-energy electrons showed that at 25 eV ssb were induced but no dsb were detected.

DNA↗
PubMed1994

Quantification of radiation-induced hydroxyl radicals within nucleohistones using a molecular fluorescent probe.

We present a method that specifically records .OH formation within histones and possibly at other sites in irradiated nucleohistone. The approach uses the radiation-induced fluorescence emissions from a chromatin-conjugated .OH detector, SECCA (a succinylated derivative of coumarin), that is converted to a fluorescent derivative, 7-hydroxy-SECCA (7-OH-SECCA), after interaction with .OH in neutral aqueous solutions. It is shown that (a) the fluorescent product 7-OH-SECCA cannot be generated by direct radiation effects after gamma or neutron irradiation of SECCA; (b) when SECCA-labeled histone is complexed with DNA to form nucleohistone, the physical properties of the modified nucleohistone are similar to those of unlabeled nucleoprotein; and (c) after irradiation of SECCA-labeled nucleohistone, a linear induction of the fluorescence signal is observed within the radiation doses examined (0.3-30 Gy). Since the sample remains available for further studies after registration of the optical signal, the current approach should permit the investigator to correlate in a single sample the localization and frequency of .OH formation with the results of other assays.

Animals↗
PubMed1993

Relative contributions of levels of initial DNA damage and repair of double strand breaks to the ionizing radiation-sensitive phenotype of the Chinese hamster cell mutant, XR-V15B. Part I. X-rays.

In order to investigate the relationships between the induction and rejoining of DNA double-strand breaks (dsb) and their biological consequences it is necessary to measure these lesions uniquely and accurately, especially at relevant low doses of ionizing radiation. Differences in radiosensitivity between cell lines could be due to variations in dsb induction or to differences in the efficiency and/or accuracy of enzymatic repair of these lesions. We have used field-inversion gel electrophoresis to investigate dsb induction and rejoining in V79B parental and XR-V15B ionizing radiation-sensitive mutant cell lines. No difference has been found in the induction of dsb in XR-V15B cells compared with wild type cells; the assay sensitivity permits measurement of dsb induced by doses as low as 1 Gy (p < or = 0.05). The radiosensitivity of the mutant cells is manifested both in a lower fraction of dsb rejoined in the early, fast repair component and longer persistence of unrejoined dsb during post-irradiation incubation. The fraction of dsb remaining unrejoined after prolonged incubations (up to 17 h) correlates well with the higher radiosensitivity of the mutant (as judged by D10 values).

Animals↗
PubMed1993

Relative contributions of levels of initial damage and repair of double-strand breaks to the ionizing radiation-sensitive phenotype of the Chinese hamster cell mutant, XR-V15B. Part II. Neutrons.

We have compared DNA double-strand break (dsb) induction and rejoining, using field-inversion gel electrophoresis, with survival in mutant (XR-V15B) and in wild-type parental (V79B) hamster cell lines after low dose neutron and X-irradiation. We found that neutrons do not appear to induce more dsbs than X-rays and deduce that increased sensitivity to neutrons is therefore not due to a higher initial yield of dsbs. Even with low doses of neutrons, there is a visible increase in the production of a smaller subset of DNA fragments which arise only after very high dose X-irradiation. In both cell lines, dsbs induced by neutrons are rejoined more slowly than those induced by X-rays. At long repair times (4 and 17 h) there are no significant differences in the fractions of unrejoined dsbs between neutrons and X-rays. We propose that neutron-induced dsbs have a higher probability of becoming lethal because they are more likely to be misrepaired during the slow stage of rejoining.

Animals↗
PubMed1993

Measurement of DNA damage by electrons with energies between 25 and 4000 eV.

All ionizing radiations deposit energy stochastically along their tracks. The resulting distribution of energies deposited in a small target such as the DNA helix leads to a corresponding spectrum in the severity of damage produced. So far, most information about the probable spectra of DNA lesion complexity has come from Monte Carlo studies which endeavour to model the relationship between the energy deposited in DNA and the damage induced. The aim of this paper is to establish methods of determining this relationship by irradiating pBR322 plasmid DNA using low energy electrons with energies comparable with the minimum energy thought to produce critical damage. The technique of agarose gel electrophoresis has been used to ascertain the fraction of DNA single- and double-strand breaks induced by monoenergetic electrons with energies as low as 25 eV. Our data show that the threshold electron energy for induction of single-strand breaks is < 25 eV, and for double-strand breaks between 25 and 50 eV.

DNA↗
PubMed1993

Field-inversion gel electrophoresis analysis of the induction and rejoining of DNA double-strand breaks in cells embedded in agarose.

Among the techniques available for the measurement of the induction and rejoining of DNA double-strand breaks (DSBs), pulsed-field gel electrophoresis appears to have the greatest potential to improve the sensitivity limits to study these lesions in the dose range closest to that used in cell survival experiments. Encapsulating the cells in agarose during the experimental procedure allows the accurate and reproducible measurement of rejoining kinetics with a very minimal time delay immediately after irradiation. The method allows direct comparison of the amount of initial DNA damage sustained with repair kinetics in experiments designed to elucidate the mechanisms underlying differences in radiosensitivity between cell lines, together with analysis of the effect of different radiation qualities. The sensitivity limits of the method are 1 Gy for the double-strand break induction experiments and 10 Gy for rejoining experiments. Under selected conditions, no significant degradation of DNA had been observed in rodent cell lines during repair incubation up to 17 h in either irradiated cells or unirradiated controls (background levels for neutron experiments, 2.2 +/- 0.3% at Time 0 compared to 2.3 +/- 0.5% after 17 h of incubation; background levels for X-ray experiments, 2.3 +/- 0.6% at Time 0 and 3.7 +/- 1.1% after 17 h of incubation). In preliminary experiments with the A549 human oat cell carcinoma cell line, DNA DSB background levels remained constant in unirradiated controls up to 4 h in the range reported for the rodent cell line.

Animals↗
PubMed1993

Evidence for induction of DNA double-strand breaks at paired radical sites.

Isolated plasmid pBR322 DNA was irradiated in the gas explosion apparatus in the presence of 10 mmol dm-3 GSH. By varying the time of the oxygen shot relative to the 5-ns pulse of electrons, the chemical repair kinetics of the oxygen-dependent free-radical precursors of DNA single- and double-strand breaks (SSBs and DSBs) can be determined. The first-order repair rate of the SSB precursors was 1370 s-1 in comparison to 2900 s-1 for DSB precursors. Under these conditions the oxygen enhancement ratio for SSBs was 3.0 in comparison to 7.5 for DSBs. This twofold difference in chemical repair rate may be interpreted on the basis of the free-radical precursor of a DSB consisting of two radicals, one on either strand of the DNA. With the chemical repair of one or other of these radicals by hydrogen atom donation from GSH, a DSB is not produced. This process will occur at twice the rate of the chemical repair of an SSB precursor consisting of a single radical. These data are consistent with the concept that DSBs are formed at the sites of clustered energy depositions with the production of a paired radical.

DNA↗
PubMed1992

Recent advances in the study of ionizing radiation damage and repair.

This workshop, organized under the auspices of the EC Concerted Action Programme on DNA Repair and Cancer, was held at the CRC Gray Laboratory, Northwood, Middlesex, UK, 23-25 October 1991. The 42 participants were drawn mainly from laboratories participating in the EC Concerted Action, with a few visitors from elsewhere. The discussions centred on the increasing convergence of classical radiobiology and biophysics with molecular biology and mammalian cell genetics to study mechanisms of DNA strand break accumulation and repair following exposure to ionizing radiation. There was a strong emphasis on the application of this research both to cancer radiotherapy and to detection of individuals at risk from cancer due to exposure to ionizing radiation. The first two days were organized as six workshop sessions; on the third day we joined forces with Julie Denekamp and dedicated a session to the memory of our late friend and colleague, Nic McNally. The rest of this day was devoted to reviews by his colleagues and collaborators of the fields of research to which he contributed so much. An evening of music and readings, organized by Joanna and Rachel McNally, completed the memorial. Here we review the first seven sessions of the workshop, emphasizing the more recent approaches and the new information they have given us.

DNA↗
PubMed1992

A comparison of the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells.

One of the important temporal stages of radiation action in cellular systems is the chemical phase, where oxygen fixation reactions compete with chemical repair reactions involving reducing agents such as GSH. Using the gas explosion technique it is possible to follow the kinetics of these fast (greater than 1 ms) reactions in intact cells. We have compared the chemical repair kinetics of the oxygen-dependent free radical precursors leading to DNA single-strand and double-strand breaks, measured using filter elution techniques, with those leading to cell killing in V79 cells. The chemical repair rates for DNA dsb (670s-1 at pH 7.2 and 380s-1 at pH 9.6) and cell killing (530s-1) were similar. This is in agreement with the important role of DNA dsb in radiation induced cell lethality. The rate for DNA ssb precursors was significantly slower (210s-1). The difference in rate between DNA ssb and dsb precursors may be explained on the basis of a dsb free radical precursor consisting of a paired radical, one radical on each strand. The instantaneous probability of one or other of these radicals being chemically repaired and not proceeding to form a dsb will be twice that of ssb radical precursor. This agrees well with the concept of locally multiply damaged sites (LMDS) produced from clusters of ionizations in DNA (Ward 1985).

Animals↗
PubMed1992

The role of non-protein sulphydryls in determining the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells.

Chinese hamster V79 fibroblasts were irradiated in the gas explosion apparatus and the chemical repair rates of the oxygen-dependent free radical precursors of DNA double-strand breaks (dsb) and lethal lesions measured using filter elution (pH 9.6) and a clonogenic assay. Depletion of cellular GSH levels, from 4.16 fmol/cell to 0.05 fmol/cell, by treatment with buthionine sulphoximine (50 mumol dm-3; 18 h), led to sensitization as regards DNA dsb induction and cell killing. This was evident at all time settings but was particularly pronounced when the oxygen shot was given 1 ms after the irradiation pulse. A detailed analysis of the chemical repair kinetics showed that depletion of GSH led to a reduction in the first-order rate constant for dsb precursors from 385 s-1 to 144 s-1, and for lethal lesion precursors from 533 s-1 to 165 s-1. This is generally consistent with the role of GSH in the repair-fixation model of radiation damage at the critical DNA lesions. However, the reduction in chemical repair rate was not proportional to the severe thiol depletion (down to approximately 1% for GSH) and a residual repair capacity remained (approximately 30%). This was found not to be due to compartmentalization of residual GSH in the nucleus, as the repair rate for dsb precursors in isolated nuclei, washed virtually free of GSH, was identical to that found in GSH-depleted cells (144 s-1), also the OER remained substantially above unity. This suggests that other reducing agents may have a role to play in the chemical repair of oxygen-dependent damage. One possible candidate is the significant level of protein sulphydryls present in isolated nuclei.

Animals↗