PubMed Health⌕ Search

Biomedical subjects

J F Ward

Publications and source records attributed to J F Ward.

At least 55 records · Page 3Linked to original sources
PubMed1996

Polyamine-induced compaction and aggregation of DNA--a major factor in radioprotection of chromatin under physiological conditions.

Spermine at physiological levels and ionic strength induces compaction and aggregation of SV40 DNA and minichromosomes resulting in marked radioprotection of the DNA against gamma-ray-induced formation of single-strand breaks. This phenomenon, termed the PICA effect, results in yields of single-strand breaks in DNA and minichromosomes comparable to those found with intact cells and is considered to be a major mechanism responsible for radioprotection of cellular DNA.

Animals↗
PubMed1996

Transport of aminothiol radioprotectors into mammalian cells: passive diffusion versus mediated uptake.

Water:n-octanol partition coefficients (KD) were determined for a series of radioprotective thiols to ascertain whether these could be used to estimate reliably their rates of uptake into mammalian cells by passive diffusion. Values of KD determined for thiols in 0.1 M potassium phosphate, pH 7.4, at 22 degrees C were: N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, WRSH), 2.0 x 10(3); dithiothreitol, 1.4; 2-mercaptoethanol, 1.7; cysteamine, 180; 3-mercaptopropanoic acid, 450; mercaptosuccinic acid, 5 x 10(6) (extrapolated value). Predictions of uptake rates by passive diffusion into mammalian cells using these values and values for the membrane diffusion rate derived from empirical evaluation of appropriate values from the literature for erythrocyte permeability paralleled the experimental rates for WR-1065 and dithiothreitol but were about threefold lower. Although the utility of KD values for quantitative prediction of uptake rates is limited, the analysis clearly indicated that uptake of aminothiols having three or more ionized amino groups will not occur at useful rates by passive diffusion. Studies of WR-1065 import by Chinese hamster V79-171 cells at micromolar levels of WR-1065 revealed an uptake that could not be explained by passive diffusion. This uptake was not inhibited by substrates for common amino acid transport systems but was inhibited by polyamines and by 1 mM DTT, which suggested that WR-33278 (WRSSWR) formed by oxidation of WRSH was being transported by a polyamine transport system. This was confirmed by showing that WRSSWR is imported efficiently by V79-171 cells treated with D,L-2-difluoromethylornithine to deplete intracellular polyamines and hence enhance their transport. Spermine inhibited uptake of WRSSWR and WRSSWR inhibited uptake of [14C]spermine, confirming that a common system is involved in the uptake of these similar molecules, both having +4 charge. It was shown that after import WRSSWR is reduced to WRSH and that uptake at low micro-molar concentrations of WRSSWR results in marked cellular concentration of the drug. These results indicate that the spermidine/spermine transport system may also provide a feasible route for import of radioprotective aminothiols bearing net charges of +3 or +4 into mammalian cells.

Animals↗
PubMed1996

Binding of radioprotective thiols and disulfides in Chinese hamster V79 cell nuclei.

Binding of thiols of varying charge (Z) in nuclei prepared in suspension was determined to assess the extent to which histones, Mg2+ spermine and chromatin structure influence counter-ion condensation of cationic thiols and co-ion depletion of anionic thiols at DNA. The nuclei were prepared in suspension buffer, washed and incubated in buffer containing thiol and graded amounts of Mg2+ and spermine. The nuclei were separated from the incubation medium by centrifugation through silicone oil, and the thiols were determined in the nuclear pellet and in the incubation buffer by labeling with monobromobimane and HPLC. Measurements of the water content of nuclei indicated that chromatin was fully condensed in buffer containing 5 mM MgCl2 and 115 mM KCl. Under these conditions nuclei incubated in 1 mM substrate had concentrations of 0.80 +/- 0.21 mM glutathione (Z = -1), 1.05 +/- 0.12 mM 2-mercaptoethanol (Z = 0), 0.95 +/- 0.15 mM cysteine (Z = 0), 0.75 +/- 0.29 mM cysteamine (Z = +1), 2.5 +/- 0.3 mM WR-1065 (Z = +2), 3.4 +/- 0.5 mM WR-35980 (Z = +3) and 12 +/- 2 mM WR-33278 (disulfide of WR-1065, Z = +4), respectively. Spermine up to 1 mM in the presence of 5 mM Mg2+ had little effect upon the binding of these thiols and disulfide, but did suppress the binding of 0.1 mM WR-33278, the results indicating that WR-33278 and spermine compete for the same sites with comparable affinity. From the results observed and the assumption that deviations from the bulk solution concentration (1 mM) result from counter-ion condensation within 3 nm of DNA, we estimate that WR-1065 (Z = +2), WR-35980 (Z = +3) and WR-33278 (Z = +4) were concentrated near DNA 6-, 8- and 20-fold, respectively, in the presence of histones, 5 mM Mg2+ and 1.0 mM spermine.

Animals↗
PubMed1996

Characterization of the reaction rate coefficient of DNA with the hydroxyl radical.

Using agarose gel electrophoresis, we have measured the yield of single-strand breaks (SSBs) induced by 137Cs gamma irradiation in a variety of plasmid DNA substrates ranging in size from 2.7 kb to 38 kb irradiated in aerobic aqueous solution in the presence of the hydroxyl radical scavenger dimethyl sulfoxide (DMSO). Under these conditions DNA SSBs are caused mainly by the hydroxyl radical. Using the competition between DMSO and DNA for the hydroxyl radical, we have estimated the rate coefficient for the reaction of the hydroxyl radical with DNA. The results cannot be characterized by conventional steady-state competition kinetics. However, it is possible to describe the second-order rate constant for the reaction as a function of the scavenging capacity of the solution. The second-order rate constant increases with increasing scavenging capacity, rising from about 5 x 10(8) dm3 mol-1 s-1 at 10(5) s-1 to about 10(10) dm3 mol-1 s-1 at 10(10) s-1. This dependence of the second-order rate constant on the scavenging capacity appears to be more pronounced for larger plasmids.

DNA↗
PubMed1996

Methylperoxyl radicals as intermediates in the damage to DNA irradiated in aqueous dimethyl sulfoxide with gamma rays.

Using agarose gel electrophoresis, we have measured the yields of DNA single-strand breaks (SSBs) for plasmid DNA gamma-irradiated in aerobic aqueous solution. Incubation after irradiation with the base damage repair endonucleases formamidopyrimidine-DNA N-glycosylase (FPG) or endonuclease III (endo III) results in an increase in the yield of SSBs. In the absence of dimethyl sulfoxide (DMSO) during irradiation, this increase is consistent with the yields of known substrates for FPG and endo III as determined by gas chromatography/mass spectrometry. After irradiation in the presence of 1 mol dm-3 DMSO, the increase in the yield of SSBs after enzyme incubation was further enhanced by a factor of about 5 to 7. The magnitude of this effect, the inability of acrylamide or oxygen to suppress it, and its attenuation by N,N,N',N'-tetramethylphenylenediamine (TMPD) or glycerol all suggest that the methylperoxyl radical (derived from DMSO) is involved as an intermediate. Reactions of the methylperoxyl radical (or some other species derived from it) do not result in strand break damage, but are responsible for DNA base damages which are recognized by FPG and endo III.

Buffers↗
PubMed1995

Mechanisms of radiosensitization in iododeoxyuridine-substituted cells.

The radiosensitization caused by iododeoxyuridine (IdU)-substitution of thymidine in V79-171 cells is decreased by the presence of acetone during irradiation. Acetone, at 1 mol dm-3, removes almost all the increase in double strand breaks (dsbs) caused by IdU substitution, but removes only about two-thirds of the enhancement in killing. Similar observations were made with BrdU-substituted cells. The decrease in cell radiosensitization coincides with the removal of the additional dsbs. The protection afforded by acetone is assumed to be due to its scavenging of hydrated electrons, thought to be the active species causing enhanced DNA damage in the presence of halogenated pyrimidines. The residual component of IdU radiosensitization, which could not be removed by treatment with acetone, is manifest largely as a shoulder effect (Dq) and may be due to either a subset of non-scavengable, lethal dsbs and/or the influence of IdU on the fixation of potentially lethal damage. This study further demonstrates that halogenated pyrimidine-mediated radiosensitization consists of at least distinct components each associated with a different phenomenon.

Acetone↗
PubMed1995

Photochemical production of double-strand breaks in cellular DNA.

In a recent publication we described a novel route for the introduction of DNA double-strand breaks (DSBs) into cellular DNA. This involved the labelling of cellular DNA with bromodeoxyuridine (BrdU) and exposure to UVA light in the presence of Hoechst dye No. 33258. Here, we report an extension of that work to the use of iododeoxyuridine (IdU); cells substituted with known levels of IdU were subjected to a similar photolysis treatment and analyzed for strand breaks by elution assays. Results indicate that both single-strand breaks (SSBs) and DSBs depend linearly on the level of IdU substitution and fluence of UVA light. The yields of SSBs and DSBs were found to be 3.5 x 10(-5) and 9.5 x 10(-7)/IdU moiety/kJm-2, respectively. These results indicate that approximately 15-fold less SSBs and 5-fold less DSBs are produced per IdU than per BrdU moiety.

Animals↗
PubMed1995

DNA repair by thiols in air shows two radicals make a double-strand break.

Using agarose gel electrophoresis, we have measured the yields of DNA single- and double-strand breaks (SSBs and DSBs) for plasmid DNA gamma-irradiated in aerobic aqueous solution. The presence during irradiation of either of the thiols cysteamine or N-(2-thioethyl)-1,3-diaminopropane (WR-1065) resulted in a concentration-dependent decrease in the yield of SSBs and a much greater decrease in the yield of DSBs. This large differential protective effect was not produced by thioethers or an alcohol of structural similarity to the two thiols, suggesting that repair of DSB radical precursors by thiols is more efficient than for SSB precursors. These observations suggest the existence of a diradical intermediate in the formation of DSBs. The results argue against a major contribution by a single radical mechanism involving interstrand radical transfer via hydrogen abstraction by a peroxyl intermediate, since the half-life of this radical transfer reaction appears to be significantly greater than the lifetime of the intermediate.

Cysteamine↗
PubMed1995

Radiation mutagenesis: the initial DNA lesions responsible.

This paper is an attempt to initiate a discussion about the causes of radiation-induced mutagenesis. It considers the evidence for the involvement of singly base-damaged sites as sources of this mutagenesis. Several sets of published data are examined: These include endogenous levels of oxidized bases and their production by natural oxidative processes, and yields of mutants produced by hydrogen peroxide and by high-LET radiation. These considerations lead to the conclusion that, at the levels at which they are produced by ionizing radiation, DNA base-damaged sites are not a cause of radiation-induced mutagenesis. In analogy with previous work we invoke the involvement of multiply damaged sites (such as double-strand breaks) produced by radiation as the causative lesions.

Animals↗
PubMed1994

Track structure, chromosome geometry and chromosome aberrations.

The joint role of radiation track structure and chromosome geometry in determining yields of chromosome aberrations is discussed. Ideally, the geometric models of chromosomes used for analyzing aberration yields should have the same degree of realism as track structure models. However, observed chromosome aberrations are produced by processes on comparatively large scales, e.g., misrepair involving two DSB located on different chromosomes or two DSB separated by millions of base pairs on one chromosome, and quantitative models for chromatin on such large scales have to date almost never been attempted. We survey some recent data on large-scale chromosome geometry, mainly results obtained with fluorescence in situ hybridization ("chromosome painting") techniques. Using two chromosome models suggested by the data, we interpret the relative yields, at low and high LET, of inter-chromosomal aberrations compared to intra-chromosomal, inter-arm aberrations. The models consider each chromosome confined within its own "chromosome localization sphere," either as a random cloud of points in one model or as a confined Gaussian polymer in the other. In agreement with other approaches, our results indicate that at any given time during the G0/G1 part of the cell cycle a chromosome is largely confined to a sub-volume comprising less than 10% of the volume of the cell nucleus. The possible significance of the ratio of inter-chromosomal aberrations to intra-chromosomal, inter-arm aberrations as an indicator of previous exposure to high LET radiation is outlined.

Cell Cycle↗
PubMed1994

The complexity of DNA damage: relevance to biological consequences.

Ionizing radiation causes both singly and multiply damaged sites in DNA when the range of radical migration is limited by the presence of hydroxyl radical scavengers (e.g. within cells). Multiply damaged sites are considered to be more biologically relevant because of the challenges they present to cellular repair mechanisms. These sites occur in the form of DNA double-strand breaks (dsb) but also as other multiple damages that can be converted to dsb during attempted repair. The presence of a dsb can lead to loss of base sequence information and/or can permit the two ends of a break to separate and rejoin with the wrong partner. (Multiply damaged sites may also be the biologically relevant type of damage caused by other agents, such as UVA, B and/or C light, and some antitumour antibiotics.) The quantitative data available from radiation studies of DNA are shown to support the proposed mechanisms for the production of complex damage in cellular DNA, i.e. via scavengable and non-scavengable mechanisms. The yields of complex damages can in turn be used to support the conclusion that cellular mutations are a consequence of the presence of these damages within a gene. Literature data are used to support these statements and to develop overall mechanisms connecting the production of primary species to the production of biologically relevant damages. The consequences of the LET of the radiation on multiplicity of damage are discussed and suggestions made for the cause of the decrease of the oxygen enhancement ratio as the LET increases.

DNA Damage↗
PubMed1994

A comparison of DNA damages produced under conditions of direct and indirect action of radiation.

Studies using a 32P-postlabelling assay reveal that at both low and high concentrations of hydroxyl radical scavenger, DNA damage from alpha-irradiation is similar to that from gamma-irradiation. At low concentrations of scavenger the identified damages are predominantly thymine glycol (Tg) and phosphoglycolate (pg) lesions, indicative of indirect hydroxyl radical (.OH) action. However, at high concentrations of scavenger (i.e. where direct effects are expected to dominate) a somewhat different pattern of damage is observed after alpha- and gamma-irradiation, with several novel lesions detected. Further experiments, in which the results of DMSO scavenging of irradiated DNA solutions are compared with glycerol scavenging and irradiation of 'dry' solid DNA, demonstrate that both direct effects and scavenger-derived secondary radicals contribute to the different spectra of lesions observed upon irradiation of highly scavenged DNA systems. Generally, irradiation under conditions that favour direct damage result in a lower relative yield of pg, whilst for the highly scavenged systems Tg damages persist. To account for this we propose that Tg are efficiently produced by direct action in these systems. Differences seen upon irradiation of the 'dry' system, compared with those of dilute aqueous solution, suggest that direct action yields a distinct spectrum of lesions.

Alpha Particles↗
PubMed1994

Response of bromodeoxyuridine-substituted Chinese hamster cells to UVA light exposure in the presence of Hoechst dye #33258: survival and DNA repair studies.

Previous work has established that DNA double-strand breaks (DSBs) are formed when Chinese hamster cells are substituted with 5-bromo-2'-deoxyuridine (BrdU) and exposed to UVA light in the presence of Hoechst dye #33258. Double-strand breaks produced by this treatment (5.1 x 10(-6) DSBs/BrdU residue/kJ m-2) were found to depend linearly on the level of BrdU substitution, Hoechst dye and fluence of UVA light. To examine the biological consequences of these novel DSBs, clonogenic assays were used to score cell survival, and elution assays were used to measure strand break levels at various times after photolysis. Using this system, marked cell killing was observed; photosensitivity could be increased by four orders of magnitude compared to cells without BrdU and dye. Decreases in the F0 value and the shoulder of survival curves followed increasing levels of BrdU substitution. In addition, the results indicate that DSBs produced by this photolysis protocol are two to three times more effective in causing cell killing than the DSBs produced by the action of ionizing radiation. To investigate the cause of the toxicity, repair of DSBs after photolysis was measured. Unexpectedly, DSB levels increased two- to threefold over 1 h at 37 degrees C, then decreased to initial damage levels over the next 2 h. The implications of this break induction are discussed in terms of mechanism and cell killing.

Animals↗
PubMed1994

DNA damage in bromodeoxyuridine substituted SV40 DNA and minichromosomes following UVA irradiation in the presence of Hoechst dye 33258.

Bromodeoxyuridine-substituted SV40 DNA and SV40 minichromosomes were prepared from infected CV1 cells, and exposed to UVA light in the presence of different concentrations of Hoechst dye 33258. Following agarose gel electrophoresis, the yields of ssbs and dsbs were determined. In DNA these yields are dependent on the level of BrdU substitution (0.038 ssb and 0.00022 dsb per BrdU residue/kJm-2), and on the mode of dye binding (type I versus II). The yields of both ssbs and dsbs were found to be lower by factors of 1.25 and 2 respectively in minichromosomes. UVA irradiation in the presence of the thiol cysteamine results in lower strand break yields, presumably due to the repair of DNA radicals formed during photolysis. The thiol was found to react seven times more slowly with the DNA radicals than oxygen. The implications of these results are discussed in light of prior cellular studies.

Bisbenzimidazole↗
PubMed1994

A charge-coupled-device camera image analysis system for quantifying DNA distributions in agarose gels after pulsed-field gel electrophoresis.

A charge-coupled-device camera system was coupled to a personal computer and, with uniformity in illumination and detection (within 4-8%) along each lane, was used for quantifying the distribution of DNA molecules that migrate from the PFGE well (plug) into the lane at distances varying from 1 to 50 mm (with 0.5 mm/pixel). By using a specially designed transmission filter for transmitting 470-725 nm fluorescence from ethidium bromide-stained DNA while eliminating most of the fluorescence (< 400 nm) from the agarose gel, and by using neutral density filters to prevent saturation of the camera, the fluorescence intensity is linearly related to the amount of DNA varying from approximately 0.03 micrograms in a 3-mm-diameter cylindrical plug 5 mm long (equal to background) to approximately 4 micrograms (where ethidium bromide staining saturates). The percentage DNA released from the plug and distribution in the lane (with 1-2 mm resolution) obtained by quantifying DNA fluorescence were not significantly different from the same data obtained by analysis of radioactivity of the same DNA labeled with [3H]dThd. However, scattering of fluorescence from one lane into an adjacent lane 3 mm away and as far as 10 mm from the plug into the lane presented a problem. This problem was overcome by using a form with slots to cover every other lane when the images were obtained and either (1) cutting the lane from the plug and moving it 15 mm away or (2) imaging the intact gel and applying a correction for approximately 7% of the fluorescence from the plug tailing out approximately 10 mm beyond the first 1 mm in the lane. In addition, the following were required: (1) carefully controlled staining and destaining procedures, and (2) a low background that is obtained as an average uniform background in each lane 5 mm beyond where DNA migration stops.

DNA↗
PubMed1994

Yield of single-strand breaks due to attack on DNA by scavenger-derived radicals.

We have measured the yield of single-strand breaks (SSBs) in plasmid DNA after 137Cs gamma irradiation, in the presence of dimethyl sulfoxide (DMSO). In the presence of oxygen, the formation of SSBs is due to hydroxyl radical attack. As the DMSO concentration is increased from 10(-4) mol dm-3 to 1 mol dm-3, the SSB yield in the presence and absence of oxygen decreases by over 100-fold and less than 10-fold, respectively. From the DMSO and DNA concentration dependencies of the SSB yield in the absence of oxygen, the second-order rate constant for the reaction of the methyl radical (derived from DMSO) and DNA can be estimated as k2 = 8.8 x 10(4) dm3 mol-1 s-1. Several other scavengers were compared with DMSO under anoxia. Radicals derived from isopropyl alcohol and glycerol also caused SSB formation in DNA, while those from 2-deoxyribose, thymine, 1,3-dimethylthymine and 1,3-dimethyluracil did not. In the case of the scavenger tert-butyl alcohol, it is unclear whether the hydrogen atom (H.) or an organic radical is responsible for the higher SSB yield under anoxic conditions.

DNA Damage↗
PubMed1994

DNA damage as the cause of ionizing radiation-induced gene activation.

Tyrosine kinases have been shown to be activated by both UVA and UVC light and gamma irradiation. For UV exposure, a non-DNA target has been proposed. Here the possibility of a non-DNA target for ionizing radiation is examined. A comparison of the fluence/dose to which the cells were exposed to elicit the response indicates that three orders of magnitude greater numbers of potentially damaging events occur after UVC exposure than after gamma irradiation. Thus, while the involvement of a non-DNA target by UVC is possible, the probability of "hits" on such targets by ionizing radiation events is remote. The levels of intracellular endogenous oxidation damage are compared to those produced by ionizing radiation. This indicates that an oxidation product which is rapidly repaired and which is therefore present at low endogenous levels, such as the DNA single-strand break, is a candidate lesion for signal induction.

Animals↗