Yields of radiation-induced main chain scission of poly U in aqueous solution: strand break formation via base radicals

The G values for single-strand breaks G(ssb) in polyuridylic acid (poly U) have been measured by low-angle laser light scattering in aqueous solutions under various conditions (e.g. in the presence of N2O, Ar and t-butanol). In N2O-saturated solutions at room temperature and pH 5.6, the G(ssb) is 2.3. The efficiency of ssb formation was found to be 41 per cent for OH radicals, 19 per cent for H atoms and congruent to zero for e-aq. On the basis of 20 per cent and less than 5 per cent attack on the sugar moiety by OH radicals and H atoms, respectively, the large G(ssb) values obtained cannot be explained solely as resulting from radicals produced by reaction of OH radicals and H atoms on the sugar moiety. It is therefore proposed that base radicals produced by the reaction of OH radicals or H atoms with the uracil moiety can also lead to chain break formation in poly U via radical transfer to the sugar moiety.     

Cell inactivation and DNA single- and double-strand breaks in cultured mammalian cells irradiated by a thermal neutron beam

The effects on the cellular viability and induction and repair kinetics of DNA strand breaks in HeLa cells were examined after exposure to a thermal neutron beam and compared with those after gamma-irradiation. The thermal neutron survival curve had no initial shoulder. The relative biological effectiveness (r.b.e.) value of the neutron beam was determined to be 2.2 for cell killing (ratio of D0 values), 1.8 and 0.89 for single strand breakage (ssb) by alkaline sedimentation and alkaline elution respectively, and for double strand breakage (dsb) 2.6 by neutral elution. No difference was observed between thermal neutrons and gamma-rays in the repair kinetics of ssb and dsb. It is suggested that the effect induced by the intracellular nuclear reaction, 14N(n,p)14C is mainly responsible for the high r.b.e. values observed.     

The influence of oxygen on the induction of radiation damage in DNA in mammalian cells after sensitization by intracellular glutathione depletion

Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and non-protein-bound SH (NPSH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10-20 per cent for the three types of radiation damage. Also under severely hypoxic conditions (0.01 microM oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0-30 per cent). At somewhat higher concentrations of oxygen (0.5-10 microM) however, the sensitization amounted to about 90 per cent for the induction of ssb and dsb and about 50 per cent for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen.     

Chromatin decondensed by acetylation shows an elevated radiation response

V-79 Chinese hamster lung fibroblasts exposed to 5 mM n-sodium butyrate were irradiated with 60Co gamma rays and cell survival was determined by the cell colony assay. In a separate set of experiments the acetylated chromatin obtained from these cells was irradiated and the change of molecular weight of the DNA was evaluated by alkaline sucrose density centrifugation. At a survival level of 10(-2) to 10(-4) cells exposed to butyrate were found to be 1.3-1.4 times more radiosensitive than control cells. Exposure of isolated chromatin to 100 Gy of 60Co gamma irradiation generated 0.9 +/- 0.03 single-strand breaks (ssb) per 10 Gy per 10(8) Da and 2.0 +/- 0.3 ssb/10 Gy/10(8) Da for control and acetylated chromatin, respectively. The elevated radiation sensitivity of chromatin relaxed by acetylation is in good agreement with previous results on chromatin expanded by histone H1 depletion [Heussen et al., Radiat. Res. 110, 84-94 (1987)]. Packing and accessibility of DNA in chromatin appear to be major factors which influence the radiation sensitivity. The intrinsic radiation sensitivity of chromatin in various packing states is discussed in light of the variation of radiation sensitivity of whole cells in the cell cycle which incorporates repair.     

D2O inhibition of interphase thymocyte death]

Using the method of flow cytometry and biochemical analysis it was shown that D2O, an agent that stabilizes microtubules, prevented the internucleosome fragmentation of DNA in thymocytes exposed to gamma radiation and dexamethasone in vitro. It was also found that D2O is ineffective with respect to Ca2+/Mg2(+)-dependent nuclease. The transfer of irradiated cells from a medium containing 90% of D2O to a normal one caused rapid DNA degradation; the fragmentation process ceased with the irradiated cells being transferred from H2O to heavy water. The results obtained permit us to assume that the disturbance of microtubules is not a trigger mechanism of DNA degradation by apoptosis, but is some intermediate stage of cell death preceding the chromatin fragmentation proper.     

DNA strand break and rejoining in cultured human fibroblasts exposed to fast neutrons or gamma rays

The production and rejoining of DNA single-strand and double-strand breaks have been monitored in monolayer cultures of proliferating human skin fibroblasts by means of sensitive techniques. Cells were irradiated with low doses of either 60Co gamma-rays or 14.6 MeV neutrons at 0 degrees C (0-5 Gy for measurement of single-strand breaks by alkaline elution and 0-50 Gy for double-strand breaks measured by neutral elution). The yield of single-strand breaks induced by neutrons was 30 per cent of that produced by the same dose of gamma-rays; whilst in the induction of double-strand breaks neutrons were 1.6 times as effective as gamma-rays. Upon post-irradiation incubation of cells at 37 degrees C, neutron-induced single-strand and double-strand breaks were rejoined with a similar time-course to gamma-induced breaks. Rejoining followed biphasic kinetics; of the single-strand breaks, 50 per cent disappeared within 2 min after gamma-rays and 6-10 min after neutrons. Fifty per cent of the double-strand breaks disappeared within 10 min, after gamma-rays and neutrons. Cells derived from patients suffering from ataxia-telangiectasia showed the same capacity for repair of single- and double-strand breaks induced by 14.6 MeV neutrons, as cells established from normal donors. The comparison of neutrons and gamma-rays in the induction of DNA breaks did not explain the elevated r.b.e. on high LET radiation. However, a study of the variation in the spectrum of lesions induced by different radiation sources will probably contribute to the clarification of the relative importance of other radio products.     

Immediate and delayed effects of low doses of beta-radiation in mammalian cells in culture

We have carried out the comparative examination into the efficacy of induction of NO and superoxide anion by incorporated and unincorporated sources of ionizing radiation in endotheliocytes (line ECV 304) and carcinoma cells (line HeLa G63) expressing various forms of NO-synthases. The increased intracellular nitric oxide levels were observed after exposure of the cells to beta-particles of 3H-thymidine and 3H2O, as well as to gamma-rays of 137Cs in HeLa G63 cells expressing the inducible forms of NO-synthases. A higher incidence of the intracellular NO level was observed after exposure to beta-particles of 3H2O than to beta-particles of 3H-thymidine or gamma-rays of 137Cs even though 3H-thymidine and gamma-rays elicited more chromosomal damages. Modification of the intracellular superoxide level was shown to have a similar dynamics of the changes in time for the both cellular lines. Shortly after irradiation, the intracellular superoxide level was lower than in non-irradiated cells, and then it became higher than the control level. The increased intracellular superoxide and NO levels were observed after exposure of the cells to beta-particles of 3H-thymidine and 3H2O, as well as to gamma-rays of 137Cs in the progeny of irradiated cells. Modification of the intracellular superoxide level was accompanied by decondensation of the cellular chromatin. A higher intracellular free radical level in the progeny of irradiated cells along with decondensation of cellular chromatin, as well as the absence of correlation between a radiation-induced structural damage of chromosomes and intracellular free radical level allow us to speculate in favor of the participation of epigenetic inheritance mechanisms.     

Difference in DNA strand break by gamma- and beta-irradiations: an in vitro study

Lambda DNA (125 micrograms/ml in Tris buffer, pH 7.4) was irradiated with 60Co gamma-rays and 3H beta-rays, respectively, and the number of strand breaks was determined by electrophoresis. Number of single-strand breaks increased linearly with radiation dose in both gamma- and beta-radiations and the relative effectiveness (beta/gamma) was found to be 1.82 in N2 and 1.16 in O2. Number of double-strand breaks increased with the square of the radiation dose in gamma-irradiation, but it increased linearly with radiation dose in beta-irradiation. Therefore, the relative effectiveness (beta/gamma) is higher at lower doses. O2 effects was observed by gamma-irradiation but was minimal after beta-irradiation.     

DNA-protein cross-linking between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells.

Formation of DNA-protein cross-links between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells is reported. Chromatin was isolated from cells, and subsequently hydrolyzed and derivatized. Analysis of derivatized hydrolysates by gas chromatography/mass spectrometry with selected-ion monitoring showed that 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]-L-tyrosine (Thy-Tyr cross-link) was formed. The presence of this DNA-protein cross-link in control cells was also observed at a level of approximately 7 molecules per 10(6) DNA nucleotides. Exposure of cells to ionizing radiation at doses between 8.7 and 82 Gy (J.kg-1) increased the amount of the Thy-Tyr cross-link linearly up to approximately fourfold over the background level. At doses higher than 82 Gy, the yield approached a plateau. Treatment of cells with H2O2 (0.5 to 10 mM) also increased the amount of the Thy-Tyr cross-link in a concentration-dependent manner. Addition of dimethyl sulfoxide and o-phenanthroline in the culture medium afforded partial inhibition of cross-link formation. Addition of catalase inhibitor KCN prior to H2O2 treatment increased the yield of cross-linking over the level observed with H2O2 treatment alone. Pretreatment of cells with ascorbic acid for 24 h without H2O2 caused formation of the Thy-Tyr cross-link. This DNA-protein cross-link in chromatin of cells is proposed to be formed by mechanisms involving a radical addition reaction and/or a radical-radical combination involving thymine and tyrosine radicals. Hydroxyl radical mediated by chromatin-bound metal ions is proposed to cause the formation of the Thy-Tyr cross-link in H2O2-treated cells.     

Recovery response of dividing cells in the thymus of whole-body gamma-irradiated mice.

Mice were irradiated with different doses of gamma-rays 30 min after the administration of 32P-orthophosphate. The dose-response curves determined at 72 hours after exposure showed an inflection point in the total activity present in the DNA in thymus and spleen. In the low dose-range, the dose-response curves have D0 = 55 rad (n = 2-5) for thymus and DO = 95 rad (n = 2-5) for the spleen. Thirty minutes after the administration of 32P-orthophosphate, the dividing cells from thymus were partially synchronized by the administration of 80 mg per kg body-weight hydroxyurea. At different time-intervals, the mice were irradiated with 80 rad, and the total activity of DNA was determined at 72 hours after synchronization. A significant maximum of recovery was found at 5 hours (S phase) after the administration of hydroxyurea. In similar conditions, the dose-response curves corresponding to the G1, S and M phase of the division cycle were also determined. The synchronization of dividing cells induced by hydroxyurea failed in the spleen.     

Chromosomal analysis of Chinese hamster V79 cells exposed to multiple gamma-ray fractions: induction of adaptive response to mitomycin C.

Chinese hamster V79 cells were irradiated daily with 0.3 Gy of gamma-rays 5 times per week for 12 weeks (total 18 Gy). These cells were challenged with an additional dose of 15. Gy gamma-rays or treated with 5 micrograms/ml of mitomycin C (MMC) for 2 h. In spite of the high total accumulated dose of gamma-rays, the number of chromosomal aberrations and sister-chromatid exchanges (SCEs) did not significantly increase in the preirradiated cells, as compared to control cells. If preirradiated cells were challenged with an additional 1.5 Gy of gamma-rays, an insignificant decrease in the yield of chromatid aberrations was observed. In contrast, preirradiated cells became significantly more resistant to the induction of chromosomal damage when challenged with mitomycin C. Our results suggest that multiple fractions of gamma-rays can induce the adaptive response to mitomycin C in preirradiated cells.     

Dicentric chromosome frequency analysis using slit-scan flow cytometry

Slit-scan flow cytometry (SSFCM) was used to quantify the frequency of dicentric chromosomes in human lymphoblastoid cells following gamma irradiation. In this study, cultured human cells were irradiated with 0, 0.25, 0.5, 1.0, and 2.0 Gy of 0.66 MeV gamma-rays, cultured for an additional 11 h, and treated for 5 h with colcemid. Chromosomes were then isolated, stained with propidium iodide, and analyzed using SSFCM for total fluorescence and slit-scan profile. The frequency of chromosomes having DNA contents greater than once and less than twice the DNA content of the number 1 chromosome and producing trimodal profiles was determined at each dose. This frequency was used as an estimate of the relative dicentric chromosome frequency at that dose. The estimated dicentric chromosome frequency per cell, f(D), increased with dose, D, in a linear-quadratic manner according to the relation f(D) = 4.52 x 10(-5) + 5.72 x 10(-5) D + 1.19 x 10(-4) D2.     

Gamma-radiolysis of poly(A) in aqueous solution: efficiency of strand break formation by primary water radicals

gamma-Radiation-induced single-strand break formation (ssb) in polyadenylic acid (poly(A] has been determined in Ar and N2O-saturated aqueous solution in the presence of different concentrations of t-butanol. Strand breaks were monitored by a low-angle laser light-scattering technique. The efficiencies for strand breakage caused by solvated electrons, hydrogen atoms and OH radicals have been found to be 0.25, 0.20 and 7.8 per cent, respectively. The efficiency of OH radicals depends only slightly on pH (pH 5.0, 7.5 and 9.0) and is independent of the presence of salt (0.01 mol dm-3 NaC1O4) and of the irradiation temperature (20 degrees C and 70 degrees C). The efficiency of OH for ssb formation obtained in this work with poly(A) is much smaller than that of poly(dA). This is explained by the different molecular conformations of the sugar moiety of poly(A) (3'-endo) and poly(dA) (2'-endo). With increasing t-butanol concentration more strand breaks are formed than expected from simple homogeneous competition kinetics of poly(A) and t-butanol for OH radicals. This result is considered to be due to nonhomogeneous reaction kinetics in the above-mentioned competition. The rate constants for the reaction of OH and H with poly(A) have been determined.     

The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity.

DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalysing the reverse of the reaction: NTP + (RNA)n = (RNA)n+1 + PPi where n indicates the number of nucleotide residues in the RNA molecule, to yield nucleoside triphosphates. This reaction requires the ternary complex of the polymerase with template DNA and the RNA that it has synthesized. It is now shown that methylenebis(arsonic acid) [CH2(AsO3H2)2], arsonomethylphosphonic acid (H2O3As-CH2-PO3H2) and arsonoacetic acid (H2O3As-CH2-CO2H) can replace pyrophosphate in this reaction. When they do so, the low-Mr products of the reaction prove to be nucleoside 5'-phosphates, so that the arsenical compounds endow the polymerase with an artificial exonuclease activity, an effect previously found by Rozovskaya, Chenchik, Tarusova, Bibilashvili & Khomutov [(1981) Mol. Biol. (Moscow) 15, 636-652] for phosphonoacetic acid (H2O3P-CH2-CO2H). This is explained by instability of the analogues of nucleoside triphosphates believed to be the initial products. Specificity of recognition of pyrophosphate is discussed in terms of the sites, beta and gamma, for the -PO3H2 groups of pyrophosphate that will yield P-beta and P-gamma of the nascent nucleoside triphosphate. Site gamma can accept -AsO3H2 in place of -PO3H2, but less well; site beta can accept both, and also -CO2H. We suggest that partial transfer of an Mg2+ ion from the attacking pyrophosphate to the phosphate of the internucleotide bond of the RNA may increase the nucleophilic reactivity of the pyrophosphate and the electrophilicity of the diester, so that the reaction is assisted.     

Induction and repair of DNA strand breaks in cultured mammalian cells following fast neutron irradiation.

Induction and repair of DNA breaks following irradiation with NIRS cyclotron neutrons were studied in cultured mammalian cells (L5178Y) in comparison to those following gamma-rays. The yield of the total single-strand breaks, 3'OH terminals and sites susceptible to S1 endonuclease following fast neutrons was found to be approximately 50 per cent of that following gamma-irradiation. On the other hand, the yield of double-strand breaks was slightly higher after fast neutrons than after gamma-rays. The percentage of the total single-strand breaks remaining unrejoined at 3 hours after post-irradiation incubation was found to be distinctly higher after the fast neutrons than after gamma-rays. The neutron-induced damage appears to carry a higher proportion of alkali-labile lesions compared to gamma-rays. It was concluded that the increase in the yield of double-strand breaks and of unrejoinable breaks is responsible for a high r.b.e. of the cyclotron neutrons.     

Pretreatment with oxygen species increases the resistance of mammalian cells to hydrogen peroxide and gamma-rays

Pretreatment of Chinese hamster ovary (CHO) or H4 (rat hepatoma) cells with low non-toxic doses of H2O2 or xanthine-xanthine oxidase renders the cells more resistant to the toxic effect of H2O2 and gamma-rays. This increased resistance is observed both in exponentially growing and in plateau-phase cells. Cells pretreated with xanthine-xanthine oxidase are less mutated than control cultures when challenged with ionizing radiation. The number of DNA single-strand breaks (measured by nucleoid sedimentation) induced by a high dose of gamma-rays or H2O2 is lower in cells pretreated with xanthine-xanthine oxidase compared to control cultures. However, the pretreatment does not modify the rate of DNA single-strand breaks rejoining in cells challenged with H2O2 or gamma-rays. The catalase activity is not modified in pretreated cells, but the superoxide dismutase activity is increased about 2-fold.     

Using Hoechst 33342 to target radioactivity to the cell nucleus

We have explored the use of Hoechst 33342 (H33342) to carry radioactivity to the cell nucleus. H33342 enters cells and targets DNA at adenine-thymine-rich regions of the minor groove. Considerable membrane blebbing and ruffling occur in CHO cells within minutes after its addition to the culture medium in micromolar quantities. Blue vesicles are apparent in the cell cytoplasm, and by 30 min the nuclei are stained dark blue. Upon its binding to DNA, a visible emission shift of the dye can be observed with fluorescence microscopy. We have radioiodinated (125I) H33342 and specifically irradiated nuclear DNA by incubating CHO cells with 125I-H33342 at 37 degrees C and accumulating 125I decays at -90 degrees C. At various times, the cells are thawed and assayed for survival (clonogenicity) and DSB (gamma-H2AX) formation. 125I-H33342 decay leads to a monoexponential decrease in cell survival with a D0 of 122 125I decays per cell and a linear increase in DNA DSB induction (equivalent to 15 gamma-H2AX foci/cell). Cell death is not modified by the radioprotective effects of H33342 because we use considerably lower concentrations than those that provide a slight protection against gamma radiation. We conclude that cell killing by 125I-H33342 and the induction of gamma-H2AX foci are highly correlated.     

The effect of 238Pu alpha-particles on the mouse fibroblast cell line C3H 10T1/2: characterization of source and RBE for cell survival

Considerable interest has been aroused in recent years by reports that the transforming and carcinogenic effectiveness of low doses of high LET radiations can be increased by reducing the dose rate, especially for transformation of 10T1/2 cells in vitro by fission-spectrum neutrons. We report on conditions which have been established for irradiation of 10T1/2 cells with high LET monoenergetic alpha-particles (energy of 3.2 MeV, LET of 124 keV microns-1) from 238Pu. The alpha-particle irradiator allows convenient irradiation of multiple dishes of cells at selectable high or low dose rates and temperatures. The survival curves of irradiated cells showed that the mean lethal dose of alpha-particles was 0.6 Gy and corresponded to an RBE, at high dose rates, of 7.9 at 80 per cent survival and 4.6 at 5 per cent survival, relative to 60Co gamma-rays. The mean areas of the 10T1/2 nuclei, perpendicular to the incident alpha-particles, was measured as 201 microns2, from which it follows that, on average, only one in six of the alpha-particle traversals through a cell nucleus is lethal. Under the well-characterized conditions of these experiments the event frequency of alpha-particle traversals through cell nuclei is 9.8 Gy-1.     

H2O2 as a DNA fragmenting agent in the alkaline elution interstrand crosslinking and DNA-protein crosslinking assays

A method for DNA fragmentation by H2O2 in the DNA alkaline elution procedure is described. Treatment of cell suspensions for 1 h with 100 microM H2O2 or 5 mM H2O2 at 0-1 degree C resulted in DNA breakage equivalent to doses of 300 and 3000 rad of gamma-rays, respectively. The elution profiles were reproducible and H2O2 was used for measurements of interstrand crosslinks and DNA-protein crosslinks induced in HeLa cells by mitomycin C, cis-diamminedichloroplatinum(II), and trans-diamminedichloroplatinum(II). The comparison of data obtained with the use of H2O2 and gamma-rays has shown that both methods have similar sensitivity and reproducibility.     

Glutathione requirement for the rejoining of radiation-induced DNA breaks in misonidazole-treated cells

The role of glutathione (GSH) in the rejoining of radiation-induced single-strand DNA breaks (ssb) was studied in human fibroblast cultures sensitized to radiation by a 30 min treatment with 1 mM misonidazole (MISO). Hypoxically irradiated cells, deficient in GSH, either inherently, or due to a 16 h incubation with 1 mM buthionine sulphoximine (BSO), rejoined the breaks after MISO treatment at a lower rate and to a lesser extent than did GSH-proficient cells. Without MISO treatment, the hypoxically induced ssb were rejoined in the GSH-deficient cells as effectively as in the proficient cells. It is concluded that a large proportion of the breaks which arise after hypoxic irradiation in the presence of MISO are of a different type to those which arise in the absence of the drug, and require a particular GSH-dependent, enzymatic repair system. This requirement for rejoining in hypoxically irradiated, MISO-treated cells is similar to that seen earlier in MISO-untreated, oxically irradiated cells, and suggests that the ssb induced by radiation in the presence of MISO or oxygen are of a similar nature.