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W F Blakely

Publications and source records attributed to W F Blakely.

34 records · Page 2Linked to original sources
PubMed1992

Inhibition of glutathione reductase activity by a carbamoylating nitrosourea: effect on cellular radiosensitivity.

Nitrosoureas inactivate cellular glutathione reductase. N,'N'1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosoureas (BCyNU), a nitrosourea reported to selectively inhibit glutathione reductase (GR) activity, was examined to determine if it could be used as a means to inhibit cellular levels of this enzyme in radiobiology studies. Confirmation of drug-induced inhibition of GR activity was demonstrated using a cell-free model system employing purified GR. Cellular studies with Chinese hamster V79A03 showed that BCyNU decreased cellular glutathione content concomitant with an inhibition of specific GR activity. Under relatively nontoxic conditions, cellular exposure to BCyNU (25 microM, 0.25 h) either before or after radiation treatment, increased cellular radiosensitivity with the optimum time for drug addition being immediately following radiation. At a BCyNU dosage which produced less than or equal to 5% cell toxicity, a marked decrease in radioresistance was characterized as a reduction in both Dq (24 +/- 1.5%) and Do (8 +/- 0.5%) concomitant with a 25 +/- 2% decrease in cellular glutathione reductase (GR) activity. At cytotoxic drug dosages (25 microM, 1 h; cell survival 79 +/- 7%), a marked radiosensitization manifested by a 1.25 +/- .07-fold reduction in the Dq was observed concomitant with a 49 +/- 4% decrease in GR activity. Using cells enriched in different stages of the cell cycle, BCyNU caused cell-age dependent cytotoxicity with preferential killing of cells in the radioresistant late-S-phase, a likely explanation for its radiosensitizing capabilities at high drug dosages. Data obtained at nontoxic drug dosages suggest that GR-inactivation may be an important component of cellular response to free-radical induced damage.

Animals↗
PubMed1992

Induction by H2O2 of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells.

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.

Animals↗
PubMed1991

Effect of fission-neutron dose rate on the induction of a tumor-associated antigen in human cell hybrids (HeLa X skin fibroblasts).

The HeLa X skin fibroblast human cell hybrid system has been used to study the effect of fission-neutron dose rate on the induction of neoplastic transformation. Previously published data using neutrons from the JANUS reactor at Argonne National Laboratory are compared with some preliminary data obtained on the TRIGA reactor at the Armed Forces Radiobiology Research Institute. Comparable results were obtained on both reactors, both in terms of absolute values of transformation frequency and in terms of magnitude of the inverse dose-rate effect (a factor of 2.5 to 3).

Antigens, Neoplasm↗
PubMed1990

Yields of radiation-induced base products in DNA: effects of DNA conformation and gassing conditions.

Gas chromatography-mass spectrometry with selected-ion monitoring was used to measure the yields of radiation-induced base products in aqueous solutions of native or heat-denatured DNA irradiated in the dose range 20-100 Gy. These DNA solutions were saturated with nitrous oxide, nitrogen, air or 20% oxygen in nitrous oxide during irradiation. The products measured were as follows: 5,6-dihydrothymine; 5-hydroxy-5,6-dihydrothymine; 5,6-dihydrothymine (thymine glycol); 5-hydroxy-5,6-dihydrocytosine; 5,6-dihydroxy-5,6- dihydrocytosine (cytosine glycol); 4,6-diamino-5-formamidopyrimidine; 7,8-dihydro-8-oxoadenine (8-hydroxyadenine); 2,6-diamino-4-hydroxy-5- formamidopyrimidine; and 7,8-dihydro-8-oxoguanine (8-hydroxyguanine). In oxygenated solutions, 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine and 5-hydroxy-5,6-dihydrocytosine were not formed. The yields of all products, other than 5,6-dihydrothymine, were greater in irradiated DNA samples from N2O-saturated solutions than from N2-saturated solutions. In N2-saturated solutions the yield of 8-hydroxyadenine was low and 8-hydroxyguanine was undetectable. Yields of pyrimidine products in heat-denatured DNA were greater than those in native DNA using all types of gases. However, the effects of DNA conformation on the yields of purine products were dependent on the type of gas used to saturate the irradiated DNA solutions. Yields of formamidopyrimidines were generally lower in solutions of DNA irradiated in the native than in the heat-denatured conformation. In air-saturated solutions of DNA, yields of 8-hydroxypurines were not influenced greatly by DNA conformation. In DNA solutions saturated with N2O/O2, 8-hydroxypurine formation was more favourable in the heat-denatured conformation than in the native conformation. On the other hand, in deoxygenated solutions, formation of 8-hydroxypurines was favoured in the native conformation. Data indicate that DNA conformation and the type of gas used to saturate the irradiated solutions have a profound influence on yields of base products in DNA.

Air↗
PubMed1990

Radiation-induced binding of DNA from irradiated mammalian cells to hydroxyapatite columns.

In experiments designed to measure radiation-induced DNA damage using the DNA unwinding-hydroxyapatite chromatography technique, we observed that under some experimental conditions a significant proportion of the test DNA became tightly bound to the hydroxyapatite (HA) and could not be released even with a high concentration of phosphate buffer. Approximately 5-10% of DNA from unirradiated cells binds to the HA. With increasing radiation doses in air, the fraction of bound DNA increases, reaching about 30% at about 35 Gy. The binding exhibits many of the characteristics of a radiation-induced cell lesion: the proportion of DNA retained by the HA is less when cells are irradiated under hypoxic conditions or in the presence of the thiol radioprotector dithiothreitol; and the binding decreases when an incubation period is allowed between irradiation and harvest of the cells for assay. Studies to determine the nature of the lesion responsible for the binding demonstrated that lesion production requires a component found in cells since no binding was observed with irradiated isolated DNA or nuclear matrix; the binding is not a result of the production of DNA-protein crosslinks; and the bound DNA is single-stranded, based on its sensitivity to nuclease S1. Because of the dose dependence of the binding of DNA to HA, the slopes of the dose-response curves for DNA damage determined with this assay depend on the method used to calculate the fraction of double-stranded DNA. Our demonstration that the bound DNA is single-stranded guides the choice of the method for data analysis.

Animals↗
PubMed1990

Hydrogen peroxide-induced base damage in deoxyribonucleic acid.

Aqueous solutions of calf thymus deoxyribonucleic acid (DNA) were exposed to hydrogen peroxide in the presence of air. Base products formed in DNA were identified and quantitated following acid hydrolysis and trimethylsilylation using gas chromatography-mass spectrometry. The yields of these products were dependent upon the hydrogen peroxide concentration, and increased in the following order: 8-hydroxyadenine, cytosine glycol, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine, thymine glycol, and 4,6-diamino-5-formamidopyrimidine. Previous studies have shown that these compounds are typically formed in DNA in aqueous solution by hydroxyl radicals generated by ionizing radiation. Hydrogen peroxide is thought to participate in a Fenton-like reaction with transition metals, which are readily bound to DNA in trace quantities, resulting in the production of hydroxyl radicals close to the DNA. This proposed mechanism was examined by exposing DNA to hydrogen peroxide either in the presence of a hydroxyl radical scavenger or following pretreatment of DNA with metal-ion chelators. The results indicate that trace quantities of transition metal ions can react readily with hydrogen peroxide to produce radical species. The production of radical species was monitored by determining the altered bases that resulted from the reaction between radicals and DNA. The yields of the base products were reduced by 40 to 60% with 10 mmol dm-3 of dimethyl sulfoxide. A 100-fold increase in the concentration of dimethyl sulfoxide did not result in a further reduction in hydrogen peroxide-induced base damage. DNA which was freed from bound metal ions by pretreatment with metal ion chelators followed by exhaustive dialysis was found to be an ineffective substrate for hydrogen peroxide. The yields of base products measured in this DNA were at background levels. These results support the role of metal ions bound to DNA in the site-specific formation of highly reactive radical species, most likely hydroxyl radicals, in hydrogen peroxide-induced damage to the bases in DNA.

Base Sequence↗
PubMed1989

Cell-cycle radiation response: role of intracellular factors.

We have been studying variations of radiosensitivity and endogenous cellular factors during the course of progression through the human and hamster cell cycle. After exposure to low-LET radiations, the most radiosensitive cell stages are mitosis and the G1/S interface. The increased activity of a specific antioxidant enzyme such as superoxide dismutase in G1-phase, and the variations of endogenous thiols during cell division are thought to be intracellular factors of importance to the radiation survival response. These factors may contribute to modifying the age-dependent yield of lesions or more likely, to the efficiency of the repair processes. These molecular factors have been implicated in our cellular measurements of the larger values for the radiobiological oxygen effect late in the cycle compared to earlier cell ages. Low-LET radiation also delays progression through S phase which may allow more time for repair and hence contribute to radioresistance in late-S-phase. The cytoplasmic and intranuclear milieu of the cell appears to have less significant effects on lesions produced by high-LET radiation compared to those made by low-LET radiation. High-LET radiation fails to slow progression through S phase, and there is much less repair of lesions evident at all cell ages; however, high-LET particles cause a more profound block in G2 phase than that observed after low-LET radiation. Hazards posed by the interaction of damage from sequential doses of radiations of different qualities have been evaluated and are shown to lead to a cell-cycle-dependent enhancement of radiobiological effects. A summary comparison of various cell-cycle-dependent endpoints measured with low- or high-LET radiations is given and includes a discussion of the possible additional effects introduced by microgravity.

Cell Cycle↗
PubMed1989

Quantitative measurement of radiation-induced base products in DNA using gas chromatography-mass spectrometry.

Gas chromatography-mass spectrometry with selected-ion monitoring was used to study radiation-induced damage to DNA. Quantitative analysis of modified purine and pyrimidine bases resulting from exposure to ionizing radiation using this technique is dependent upon the selection of appropriate internal standards and calibration of the mass spectrometer for its response to known quantities of the internal standards and the products of interest. The compounds 6-azathymine and 8-azaadenine were found to be suitable internal standards for quantitative measurement of base damage in DNA. For the purpose of calibration of the mass spectrometer. relative molar response factors for intense characteristic ions were determined for the trimethylsilyl derivatives of 5-hydroxyuracil, thymine glycol, and 5,6-dihydrothymine using 6-azathymine, and for the trimethylsilyl derivatives of 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine using 8-azaadenine. Accurate measurement of the yield of radiation-induced modifications to the DNA bases is also dependent upon two chemical steps in which the purines and pyrimidines are released from the sugar-phosphate backbone and then derivatized to make them volatile for gas chromatography. The completeness of these reactions, in addition to assessing the stability of the modified DNA bases in acid and their trimethylsilylated derivatives over the time necessary to complete the experimental analysis was also examined. Application of this methodology to the measurement of radiation-induced base modification in heat-denatured, nitrous oxidesaturated aqueous solutions of DNA is presented.

DNA↗
PubMed1988

Effect of DNA conformation on the hydroxyl radical-induced formation of 8,5'-cyclopurine 2'-deoxyribonucleoside residues in DNA.

Reactions of hydroxyl radicals with DNA form a variety of base and sugar products and 8,5'-cyclopurine 2'-deoxyribonucleoside residues in DNA. Here we report the effect of DNA conformation on the yields of 8,5'-cyclopurine 2'-deoxynucleosides and the ratios of their (5'R)- and (5'S)-diastereomers. Calf thymus DNA in native (double-stranded DNA) or heat-denatured form (single-stranded DNA) was exposed to hydroxyl radicals generated by ionizing radiation in nitrous oxide-saturated phosphate buffer. Doses ranging from 10 to 40 Gy were used to ensure low levels of damage to DNA and thus to preserve its secondary structure in experiments with double-stranded DNA (ds-DNA). After irradiation, DNA was hydrolysed enzymatically to 2'-deoxyribonucleosides. The hydrolysates were dried, trimethylsilylated, and analyzed by capillary gas chromatography-mass spectrometry with selected-ion monitoring. An internal standard was used for quantitative measurements and added to DNA samples prior to enzymatic hydrolysis. The yields of 8,5'-cyclo-2'-deoxyadenosine and 8,5'-cyclo-2'-deoxyguanosine in single-stranded DNA (ss-DNA) were higher than those in ds-DNA. The (5'R)-diastereomers of both compounds were found to predominate over their (5'S)-diastereomers in ss-DNA. In contrast, the yields of the (5'S)-diastereomers in ds-DNA were slightly higher than those of the (5'R)-diastereomers. The G values of 8,5'-cyclo-2'-deoxyadenosine in ss-DNA and ds-DNA were 0.042 and 0.025, respectively. Those of 8,5'-cyclo-2'-deoxyguanosine in ss-DNA and ds-DNA were 0.038 and 0.017, respectively.

DNA↗
PubMed1988

Cell-cycle dependence of X-ray oxygen effect: role of endogenous glutathione.

The oxygen effect was measured in human T-1 cell populations synchronized by mitotic selection and x-irradiated in vitro after they were allowed to progress to six different ages during the division cycle. Survival curves and dose-ratio calculations with 95% confidence intervals were obtained from computer fits of the data to the linear-quadratic model. The oxygen enhancement ratio (OER) values at the 1% survival increased level were 2.6 +/- 0.08 in G1/early S phase and increased to 3.0 +/- 0.15 in late S/G2 phase. The OER values at 10% survival increased linearly from 2.6 +/- 0.2 for G1-phase cells to 3.2 +/- 0.2 for late S/G2-phase cells. The increased OER in S-phase cells was the result of a greater hypoxic radioresistance compared with that measured with G1-phase cells. In parallel experiments with synchronized cell populations, glutathione (GSH) and glutathione disulfide levels were measured by the Tietze assay and also were found to increase over the same period. The molecular mechanisms responsible for the radiation response involve a number of factors, one of which in this cell line may be GSH levels, especially under conditions of hypoxic exposure. Our data are consistent with the hypothesis that G1- to late S-phase, age-dependent fluctuations in GSH content may be correlated with changes in OER during the human T-1 cell cycle. Changes in GSH content relative to its constitutive levels in the cell and alternative reductive factors (i.e., protein thiols), as well as their cellular location, may be important factors in the comparison of these findings to other cell lines.

Cell Cycle↗
PubMed1987

Glucan: mechanisms involved in its "radioprotective" effect.

It has generally been accepted that most biologically derived agents that are radioprotective in the hemopoietic-syndrome dose range (eg, endotoxin, Bacillus Calmette Guerin, Corynebacterium parvum, etc) exert their beneficial properties by enhancing hemopoietic recovery and hence, by regenerating the host's ability to resist life-threatening opportunistic infections. However, using glucan as a hemopoietic stimulant/radioprotectant, we have demonstrated that host resistance to opportunistic infection is enhanced in these mice even prior to the detection of significant hemopoietic regeneration. This early enhanced resistance to microbial invasion in glucan-treated irradiated mice could be correlated with enhanced and/or prolonged macrophage (but not granulocyte) function. These results suggest that early after irradiation glucan may mediate its radioprotection by enhancing resistance to microbial invasion via mechanisms not necessarily predicated on hemopoietic recovery. In addition, preliminary evidence suggests that glucan can also function as an effective free-radical scavenger. Because macrophages have been shown to selectively phagocytize and sequester glucan, the possibility that these specific cells may be protected by virtue of glucan's scavenging ability is also suggested.

5'-Nucleotidase↗
PubMed1986

Formation of cytosine glycol and 5,6-dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide.

OsO4 selectively forms thymine glycol lesions in DNA. In the past, OsO4-treated DNA has been used as a substrate in studies of DNA repair utilizing base-excision repair enzymes such as DNA glycosylases. There is, however, no information available on the chemical identity of other OsO4-induced base lesions in DNA. A complete knowledge of such DNA lesions may be of importance for repair studies. Using a methodology developed recently for characterization of oxidative base damage in DNA, we provide evidence for the formation of cytosine glycol and 5,6-dihydroxycytosine moieties, in addition to thymine glycol, in DNA on treatment with OsO4. For this purpose, samples of OsO4-treated DNA were hydrolysed with formic acid, then trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry. In addition to thymine glycol, 5-hydroxyuracil (isobarbituric acid), 5-hydroxycytosine and 5,6-dihydroxyuracil (isodialuric acid or dialuric acid) were identified in OsO4-treated DNA. It is suggested that 5-hydroxyuracil was formed by formic acid-induced deamination and dehydration of cytosine glycol, which was the actual oxidation product of the cytosine moiety in DNA. 5-Hydroxycytosine obviously resulted from dehydration of cytosine glycol, and 5,6-dihydroxyuracil from deamination of 5,6-dihydroxycytosine. This scheme was supported by the presence of 5-hydroxyuracil, uracil glycol and 5,6-dihydroxyuracil in OsO4-treated cytosine. Treatment of OsO4-treated cytosine with formic acid caused the complete conversion of uracil glycol into 5-hydroxyuracil. The implications of these findings relative to studies of DNA repair are discussed.

Barbiturates↗
PubMed1985

Mammalian cells are not killed by DNA single-strand breaks caused by hydroxyl radicals from hydrogen peroxide.

Cell killing by ionizing radiation has been shown to be caused by hydroxyl free radicals formed by water radiolysis. We have previously suggested that the killing is not caused by individual OH free radicals but by the interaction of volumes of high radical density with DNA to cause locally multiply damaged sites (LMDS) (J. F. Ward, Radiat. Res. 86, 185-195, 1985). Here we test this hypothesis using hydrogen peroxide as an alternate source of OH radicals. The route to OH production from H2O2 is expected to cause singly damaged sites rather than LMDS. Chinese hamster V79-171 cells were treated with H2O2 at varying concentrations for varying times at 0 degree C. DNA damage produced intracellularly was measured by alkaline elution and quantitated in terms of Gray-equivalent damage by comparing the rate of its elution with that of DNA from gamma-irradiated cells. The yield of DNA damage produced increases with increasing concentration of H2O2 and with time of exposure. H2O2 is efficient in producing single-strand breaks; treatment with 50 microM for 30 min produces damage equivalent to that formed by 10 Gy of gamma irradiation. In the presence of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), the yield of damage decreases with increasing DMSO concentration consistent with the scavenging of hydroxyl radicals traveling an average of 15 A prior to reacting with the DNA. In contrast to DNA damage production, cell killing by H2O2 treatment at 0 degree C is inefficient. Concentrations of 5 X 10(-2) M H2O2 for 10 min are required to produce significant cell killing; the DNA damage yield from this treatment can be calculated to be equivalent to 6000 Gy of gamma irradiation. The conclusion drawn is that individual DNA damage sites are ineffectual in killing cells. Mechanisms are suggested for killing at 0 degree C at high concentrations and for the efficient cell killing by H2O2 at 37 degrees C at much lower concentrations.

Animals↗
PubMed1984

Effects of inhibitors of DNA strand break repair on HeLa cell radiosensitivity.

The effects of three drugs (hydroxyurea, 1-beta-arabinofuranosylcytosine, and diamide) known to inhibit DNA synthesis on the repair of ionizing radiation-induced DNA single-strand breaks measured by alkaline elution and on cellular radiosensitivity were examined. Inhibition of repair was observed at 10(-2) M hydroxyurea, 10(-4) M 1-beta-D-arabinofuranosylcytosine, and 5 X 10(-5) M diamide, levels causing only 10% cell kill. While the mechanisms by which the drugs inhibit DNA synthesis differ, they are equally effective at inhibiting repair; without drug, cells, after a dose of 10 grays, repair 35% of DNA strand breaks in 3 min and a further 35% in 1 hr; with drug, only 10% is repaired in 3 min, and the deficiency in repair amount remains, even after 60 min. The effect of similar drug treatment on radiation-induced cell killing shows that radiosensitivity is increased; the major effect is reduction in D0 from 1.3 grays to approximately 0.8 grays with smaller effects on Dq. The data are consistent with the hypothesis that radiation produces potential double-strand breaks in DNA which, if not rapidly repaired, are converted into lethal actual double-strand breaks.

Azo Compounds↗