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.     

The contribution of the nuclear reaction 1H(n, gamma)2D to the yield of DNA single strand breaks in cultured mammalian cells irradiated by thermal neutrons

The yield of single strand breaks (ssb) in DNA of the HeLa S-3 cells after thermal neutron irradiation was examined using the alkaline sucrose gradient method. The contribution of the 1H(n, gamma)2D reaction to the yield of ssb was determined by substituting D2O for H2O in the irradiated medium. Calculation shows that when cells are irradiated in the H2O medium, the per cent contribution of the contaminating gamma-rays, the nuclear reaction 1H(n, gamma)2D and the other nuclear reactions is 31, 44 and 25 per cent respectively assuming additivity of effects. The estimated number of ssb induced by the nuclear reaction 1H(n, gamma)2D was at least 4.4 times greater than that by 60Co gamma-rays at the same absorbed dose. Two possible interpretations are discussed to explain the high efficiency of the 1H(n, gamma)2D reaction for ssb induction.     

Structure and optical activity of the DNA-aminoacridine complex

The electronic absorption and circular dichroism spectra of the DNA-acridine orange complex have been measured over a range of ionic strength, pH, and DNA phosphate to dye (P/D) ratios. Three circular dichroism bands associated with the long wavelength absorption band of acridine orange are induced on complex formation with DNA. Two of the dichroism bands, due mainly to dimeric dye molecules, are favored by low ionic strength, low pH (3.2), and a low P/D ratio (～3), while the third, deriving primarily from monomeric dye, is optimum at high ionic strength, neutral pH, and a larger P/D ratio (9). The data suggest that monomeric acridine orange binds to DNA in the form of a left-handed helical array with four dye molecules per turn, while the bound dimer has a skewed sandwich conformation which is itself dissymmetric. The stereochemical relations between the bound monomer dye and the DNA are consistent with a modified intercalation model for the DNA-acridine complex.     

Induction of DNA strand breaks by laser irradiation in a visible range

A possibility of formation of breaks in DNA chains under the influence of visible laser radiation (lambda = 532 nm) on DNA--dye-intercalator complexes has been demonstrated theoretically and corraborated experimentally. The most probable mechanisms of the induction of breaks in DNA chains are discussed. Theoretical estimates and the experiments have been performed on complexes of DNA of lambda-phage with acridine orange dye (AO) under irradiation of these complexes by picosecond (tau p = 30 ps) pulses of second harmonic YAG:Nd laser. A strong effect of the formation of double-strand breaks is observed at light intensity of some GW/sm2 and dose of several hundred pulses.     

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.     

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.     

DNA replication in mammalian cells after exposure to factors of a physical, chemical or biological nature. III. 5-Fluorodeoxyuridine induces DNA synthesis in cells not suppressed by gamma irradiation

The influence of preincubation of HeLa and Chinese hamster V79 cells with fluorodeoxyuridine (FUdR, 10(-6) M) on DNA replication and molecular weight of nascent DNA was studied after gamma-irradiation with a dose as much as 10 Gy. The 60Co-radiation inhibits DNA synthesis in both HeLa and V79 cells by 30-40 per cent. The incubation with FUdR before irradiation suppresses the inhibitory effect of irradiation on DNA synthesis. It is suggested that differences in gamma-radiation inhibition of DNA synthesis may result from the FUdR-induced changes in chromatin structure, rather than from synchronization of cell growth. This suggestion is based on the observation that the radioresistant mode of DNA synthesis occurred 18 hours following the short-term (6 hours) incubation with FUdR in cell cultures differing from each other in almost 2-fold their cell longevity.     

Mechanism of differential staining of BUdR-substituted Vicia faba chromosomes.

Chromosomes of the broad bean Vicia faba were isolated and air-dried on slides after incorporation of BUdR into DNA (BUdR substitution) for two rounds of replication. Then the preparations were embedded in a buffer solution containing trypsin as well as fluorescence dye (acridine orange or Hoechst 33258). We observed chromosomes with a fluorescence microscope at various times after embedding. After about 15 min one sister chromatid of some of the metaphase chromosomes showed enhanced darkening and disintegration within 1–4 min (melting effect) during observation. We suppose that fragmentation of BUdR-substituted DNA by the acridine orange-visible light system in acridine orange staining and by irradiation with wavelengths around the transition from UV to visible light in Hoechst 33258 staining is responsible for this phenomenon. The disintegration of one sister chromatid in BUdR-substituted chromosomes can also be produced by UV irradiation during trypsin treatment when fluorescence dyes are not present.     

Structural investigation of radiation-induced aggregates of ribonuclease

Following irradiation of bovine pancreatic ribonuclease in aqueous solution with 60Co gamma-rays protein aggregates are formed. The nature of the bonds linking these radiation-induced aggregates together has been investigated by chromatographic and electrophoretic methods. Thin-layer gel filtration and polyacrylamide gel electrophoresis, both in the presence of sodium dodecyl sulphate, demonstrated the existence of covalent crosslinks between the aggregates. However, non-covalent crosslinking also plays a role in the radiolysis of ribonuclease. Thin-layer gel filtration with and without 6 M urea and 2 per cent beta-mercaptoethanol added to the gel, revealed that only part of the covalent bonds between the aggregates consisted of disulphide linkages. By separation of the reduced aggregates by thin-layer gel filtration and electrophoresis, both with SDS, this finding was substantiated. Densitometric measurements indicated for example that the percentage of covalently linked dimers held together by disulphide bridges amounted to about 40-45 per cent, whereas the remaining 55-60 per cent of the dimers must be linked by other covalent bonds. The existence of covalent crosslinks other than disulphide bonds was also confirmed by isoelectric focusing. By this method definite differences were established between the proteolytic hydrolysates of the reduced aggregates and the reduced monomer of gamma-irradiated ribonuclease.     

Multifraction radiation response of mouse lung

The response of mouse lung to repeated doses of 60Co gamma-rays of as low as 115 cGy per fraction was measured using death from pneumonitis between 80 and 120 days after irradiation as the endpoint. A fractionation interval of 3 h was maintained for most regimens but in the longer experiments some 12 h intervals were introduced for logistic reasons. The longest overall duration (for a 43 fraction regimen) was 8 days. The total doses required to produce 50 per cent mortality increased continuously as dose/fraction was decreased, even from 160 to 115 cGy per fraction. Of clinical relevance, the steepness of the isoeffect curve over the dose range 115-500 cGy indicates that the lung shows greater sparing from dose fractionation than is characteristic of more rapidly-responding normal tissues, resembling, in this respect, other more slowly-responding tissues such as spinal cord. The plot of the reciprocal of the LD50 values as a function of dose per fraction was non-linear, suggesting that a linear quadratic dose response model may not be appropriate or that repair of cellular injury in lung is not complete in 3 h, or both.     

Intercalation of cationic dyes in the DNA double helix: introductory theory

The effect of salt on the intercalation of acridine dyes and DNA is rather well explained by the Gouy-Chapman double-layer theory as applied to a cylinder model of the DNA–dye complex. The free energy of transfer of a dye ion from the bulk solution to the complex is divided into several parts, one of which, ΔF₀, accounts for the short-range, nonelectrostatic interactions. The assumption that ΔF₀ should not depend on the amount of dye in the complex leads to an internal dielectric constant of the cylinder of about D_i = 7. The scatter in ΔF₀ values, as calculated from individual experimental points, is of order 0.5 kT per dye ion. This scatter is large enough to mask possible effects of heterogeneity in DNA sequences. The calculations are made for a long cylinder with radius 10 Å, with the DNA phosphate charges smeared uniformly at the surface, a uniform spacing of dye charges at the cylinder axis, and a length of b = 3.37 Å per base pair. Each intercalated dye ion also adds a length b to the total length of the cylinder. The salt-dependent part of the electric free energy of intercalation, ΔF₁, is tabulated for complexes with r = 0–0.24 dye ions per DNA phosphate in 0.002–0.2M monovalent salt and dye solutions.     

Assessment of DNA damage and repair in human peripheral blood mononuclear cells using a novel DNA unwinding technique.

A newly developed, fast and sensitive microplate assay (Fast Micromethod) was used for the assessment of gamma-radiation-induced DNA damage in peripheral blood mononuclear cells (PBMC) from healthy donors of various ages and from cancer patients undergoing radiotherapy. This assay detects the presence of DNA single-strand breaks and alkali-labile sites by monitoring the rate of DNA unwinding under alkaline conditions using the fluorescent dye PicoGreen, which preferentially binds to double-stranded DNA at high pH (>12.0); it requires only minimal amounts of material (approximately 3 x 10(3) cells/well) and can be performed within 3 hrs. or less. EDTA blood samples were collected from patients not undergoing chemotherapy prior and immediately after irradiation, or were collected from healthy donors and irradiated ex vivo. The results revealed that the amount of DNA strand breaks in PBMC, induced by application of a single dose to patients in the course of radiotherapy treatment, markedly varied between different individuals. To examine the effect of age on DNA damage, the basal levels of DNA damage in PBMC from a total of 30 healthy donors were determined: 10 were 20 to 30 years of age, 10 were 40 to 60 years of age and 10 were >70 years of age. It was found that the mean basal level of DNA damage from donors in the >70-year age group was significantly higher (by 97%) than that of the 20- to 30-year age group and 27% higher than that of the 40- to 60-year age group. Measurements of the level of induced DNA damage in PBMC isolated from blood after 2 Gy irradiation with 60Co gamma-rays revealed no significant differences between donors aged 20-30 and 40-60. However, there was a strong increase (by 2.3- to 2.9-fold) in radiosensitivity in the age group >70. The microplate assay described may be used as a pretherapeutic sensitivity test for the assessment of the individual radiosensitivity of patients prior to radiation therapy.     

Fast neutron r.b.e. for lethality and genotoxicity in a wild-type and a repair-deficient strain of yeast

The relative biological effectiveness (r.b.e.) of cyclotron-produced fast neutrons (11 MeV) in relation to 60Co gamma-rays, was studied in a wild-type and a DNA repair-deficient yeast strain for cell killing and genotoxicity. In the wild-type (D7) strain the r.b.e. varied from 2.7 to 4.1 for lethality, 2.8 to 7.1 for reverse mutation and 3.5 to 7.8 for mitotic gene conversion. At different survival levels, the repair deficient strain (D7 rad 52/rad 52) generally showed a lower r.b.e. for both cell killing and genotoxicity (25.2 to 37.2 per cent reduction for the cell death and 24.8 to 70.6 per cent for mutation and gene conversion) compared to the wild type. Except at very low dose levels, the r.b.e. values for cell killing and genotoxicity were similar within a given strain. At similar survival levels, neutrons were no more genotoxic than gamma-rays.     

The induction and repair of DNA damage detected by the DNA precipitation assay in Chinese hamster ovary cells treated with acridine orange + visible light

The photodynamic effect of the dye acridine orange (AO) in combination with visible light (400-700 nm) was studied in Chinese hamster ovary (CHO) cells, the endpoints investigated being induction, as well as repair, of DNA strand breaks. Cells were treated for 20 min with AO (0.1-3.0 micrograms/ml), washed free of excess dye and subsequently exposed to low doses of visible light (2 x 40 W/8 W/m2) for 5-15 min. AO proved to be an efficient sensitizer for light-induced DNA strand breaks, detected with the DNA precipitation assay, and expressed as percentage of DNA precipitated. The induction of breaks was linear up to 0.5 micrograms/ml AO + 10 min of light, which corresponds to 55% precipitated DNA, and was dependent on the concentration of AO as well as on the dose of light delivered. As a comparison, 18 Gy of X-rays was required to yield an equivalent amount of induced DNA strand breaks. The rejoining of the light-induced DNA strand breaks was studied by incubating the AO-sensitized cells for 30-120 min at 37 degrees C directly after light exposure. A fast recover of 67-91% of the damage (compared to initial damage, recovery time = 0, and dependent on the concentration of AO) was observed during the first 30 min of incubation. However, a significant amount of DNA damage remained after 2 h of recovery. These remaining, long-lived lesions might be involved in the photoinduced and acridine-sensitized chromosomal aberrations and sister-chromatid exchanges (SCE). The significance of these observations is discussed in relation to AO-sensitized and photoinduced DNA damage and chromosomal alterations.     

Fluorescent analysis of irradiation-induced changes in chromatin state

A fluorescent technique with Hoechst-33258 and acridine orange staining was used to assess changes in chromatin state induced by radiation. Fluorochromes with different modes of binding to DNA were chosen. In T lymphocytes chronic irradiation caused a rearrangement of binding between nonhiston proteins and lipids accompanied by conformational changes in DNA, resulting in chromatin condensation. The decrease in fluorescence probably resulted from a reduction in the number of sites accessible for dye binding. After acute irradiation, the fluorescence intensity decreases predominantly due to double-strand breaks.     

Experimental and kinetic studies on methylene blue adsorption by coir pith carbon

Varying the parameters such as agitation time, dye concentration, adsorbent dose, pH and temperature carried out the potential feasibility of thermally activated coir pith carbon prepared from coconut husk for removal of methylene blue. Greater percentage of dye was removed with decrease in the initial concentration of dye and increase in amount of adsorbent used. Kinetic study showed that the adsorption of dye on coir pith carbon was a gradual process. Lagergren first-order, second-order, intra particle diffusion model and Bangham were used to fit the experimental data. Equilibrium isotherms were analysed by Langmuir, Freundlich, Dubnin-Radushkevich, and Tempkin isotherm. The adsorption capacity was found to be 5.87 mg/g by Langmuir isotherm for the particle size 250-500 microm. The equilibrium time was found to be 30 and 60 min for 10 and 20 mg/L and 100 min for 30, 40 mg/L dye concentrations, respectively. A maximum removal of 97% was obtained at natural pH 6.9 for an adsorbent dose of 100 mg/50 mL and 100% removal was obtained for an adsorbent dose of 600 mg/50 mL of 10 mg/L dye concentration. The pH effect and desorption studies suggest that chemisorption might be the major mode of the adsorption process. The change in entropy (DeltaS0) and heat of adsorption (DeltaH0) of coir pith carbon was estimated as 117.20 J/mol/K and 30.88 kJ/mol, respectively. The high negative value of change in Gibbs free energy indicates the feasible and spontaneous adsorption of methylene blue on coir pith carbon.     

Change in the succinate dehydrogenase activity of peripheral lymphocytes in the rat in early periods following exposure to ionizing radiation

Activity of succinate dehydrogenase (SDH) in peripheral blood lymphocytes of albino mongrel rats was determined during an hour following gamma-irradiation. The highest SDH activity in blood lymphocytes (a 41 per cent increase) was registered 20-30 min following 75 Gy irradiation of the head. With a dose of 20 Gy the SDH response was twice as low. During 1 h following irradiation with a dose of 200 Gy the SDH activity exhibited a 10 per cent decrease.     

Romanowsky dyes and the Romanowsky-Giemsa effect. 4. Binding of azure B to DNA

We investigated the binding of azure B to DNA (calf thymus) over a wide range of concentrations of the dye (CF) and the nucleic acid (CN) using absorption spectroscopy [CF and CN represent the total concentrations of the ye (F) and the mononucleotide units (N) of the DNA, respectively]. The binding isotherms of the dye to DNA in aqueous solutions were determined. In addition, we analysed the composition of insoluble DNA/azure B precipitates that are formed in presence of an excess of azure B. These precipitates are of particular interest, because Giemsa staining is usually performed using high dye concentrations. Azure B easily forms dimers in aqueous solutions. When determining the binding isotherms, the equilibrium between free monomers and dimers must be taken into account. Therefore, we determined the dimerisation constant (Kd) of azure B from the concentration dependency of its absorption spectra in water at the standard temperature T = 298 K (25 degrees C), Kd = 6.5 X 10(3) M-1 (experimental conditions: tris buffer, pH 7.2; concentration of Na ions, CNa = 0.002 M). As the CNa value increases, the dimerisation constant rises rapidly. When the azure B concentration is very low and there is an excess of DNA, ordinary Scatchard and Langmuir isotherms are observed. Monomer dye cations are bound to DNA, these cations being in equilibrium with free monomers in the solution. In order to obtain the Scatchard binding constant (Ks) and the binding parameter (n) spectroscopically, it is necessary to determine the extinction coefficient (epsilon Fb) of the monomer bound (b) dye molecules (F) at one analytical wave number (upsilon a). The three constants can be determined simultaneously using an iterative technique that combines Scatchard isotherms and the Benesi-Hildebrand extrapolation, CN----infinity. We obtained Ks = 1.8 X 10(5) M-1 and n = 0.18 (25 degrees C; tris buffer, pH 7.2; CNa = 0.002 M). At very low dye (CF) and competitor (CNa) concentrations, only 18% of the anionic binding sites of the DNA are capable of binding the dye cations. With increasing CNa values the concentration of bound azure B cations decreases rapidly. The Na cations displace the bound dye cations and act as a competitor. The Ks value also greatly depends on the competitor concentration (CNa).(ABSTRACT TRUNCATED AT 400 WORDS)     

Level and nature of the drug resistance of Shigella flexneri]

Characteristics of antibiotic resistance of 300 strains of Shigella flexner 2a isolated from patients within 1976--1977 in the regions where these bacteria were very rare for a long period of time were studied. It was shown that most of the isolates were resistant to chloramphenicol (88.1 per cent), tetracycline (94.8 per cent), streptomycin (89.1 per cent), polymyxin M (82.4 per cent) and others. 46.5--61.6 per cent of the isolates were resistant to neomycin antibiotics. A high level of the resistance was also noted: the bactericidal effect was registered in 52.0-74.0 per cent of the cultures at a dose of 500--1000 microgram/ml. 91.4 per cent of the strains possessed multiple dug resistance, 78.8 per cent of them being simultaneously resistant to 4--7 drugs. Transmissive R-plasmids were found in 68.8 per cent of the isolates. After exposure to acridine dyes the plasmid nature of the resistance was confirmed in 72.3 per cent of the cultures. Variability of the r-determinant sets in r-plasmids was noted. Strains (64.9 per cent) carrying r-determinants Tc, Cm, Sm and Tc, Cm were more frequent. Strains with one transmissive r-determinant were usually solitary.     

Relaxation of supercoiled plasmid DNA by oxidative stresses in Escherichia coli.

The relaxation of plasmid DNA was observed after the visible light irradiation of Escherichia coli AB1157 harboring plasmid pBR322 or some other plasmids in the presence of a photosensitizing dye, such as toluidine blue or acridine orange, and molecular oxygen. Treatment of the cells with hydroperoxides, such as tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide, also caused the plasmid DNA relaxation in vivo. Relaxation was not observed in these treatments of purified pBR322 DNA in vitro. Plasmid DNA relaxation was also detected after near-UV irradiation. Far-UV irradiation did not induce such relaxation.