Conformational properties of DNA after exposure to gamma rays and neutrons.

DNA aqueous solutions were irradiated with 0-40 Gy of 60Co gamma rays and 0-1.5 Gy of (Pu-Be) neutrons. Thermal transition spectrophotometry (TTS) was used to trace the changes in the DNA conformation at the above doses. Previous results using the perturbed angular correlation (PAC) method were used to complement to the current analysis. The TTS and PAC methods are two different approaches to the study of the effects of radiation on DNA. Both showed that neutrons are more effective than gamma rays in inducing DNA damage. The TTS method showed that neutrons are 11 +/- 5 times more efficient than gamma rays, while the PAC method had shown this value to be 34 +/- 4. From the current study we deduced that the radiation damage to DNA is not a spontaneous effect but rather is an ensemble of damaging events that occur asynchronously. Any single method selected for the study of such damages can concentrate on only a part of the damage, leading to over- or underestimation of the relative effectiveness of the neutrons.

Changes in DNA flexibility after irradiation with gamma rays and neutrons studied with the perturbed angular correlation method.

Neutron and gamma irradiation of buffered solutions of calf thymus DNA resulted in changes in the dynamics of the macromolecule. In the low-dose region (0.8-10 cGy of 239Pu-Be neutrons and 0.34-3 Gy of 60Co gamma rays), the flexibility of DNA decreased as indicated by slower rotation of the molecules. Neutrons appeared to be approximately 35 times more effective than 60Co gamma rays. The rotational correlation time, tau C, was measured using the perturbed angular correlation (PAC) method. Its variation appears to follow a linear-exponential behavior. An attempt is made to formulate this behavior as a function of the energy deposited on the macromolecule (radiation dose), the average threshold energy (dose) required to form new lesions, and the available population of intact DNA sites.

Probing irradiated DNA with the perturbed angular correlation method.

The technique of perturbed angular correlations of gamma rays has been used to study the effects of radiation on DNA molecules. The samples are buffered solutions of calf thymus DNA exposed to various doses (0-80 Gy) of gamma rays. Indium-111 is used as a probe. Rotational correlation times, tau(c), a parameter measuring the flexibility of a macromolecule, are obtained that show a dependence on radiation dose.

Diversity of peripheral blood mononuclear cells as revealed by a novel multiple microgel "comet assay".

Multiple microgel comet assay (MMCA) is a metho-dological adaptation of the single-cell gel electrophoresis assay in which we have introduced the use of standard agarose plug molds in an attempt to improve and expand the applications of the assay. We focused on the study of the heterogeneity of peripheral blood mononuclear cells (PBMC) at the level of the basal single-strand breakage and the DNA damage induction caused by ionizing radiation. Differences among subpopulations were also investigated at the level of chromatin organization and methylation after NotI digestion of microgel-embedded cells. In parallel experiments, the NotI-digested nucleoids were also analyzed with the use of pulsed-field gel electrophoresis (PFGE) and the DNA migration patterns were compared with the corresponding patterns from the MMCA. Significant heterogeneity in the distribution of the oxidative DNA damage, as well as intracellular variations in the NotI digestion patterns were observed in the cell population of PBMC. The combined use of both the comet assay and PFGE provides a useful model for analysis of variation in DNA damage in individual cells as well as information on size of DNA fragments.

Effects of radical scavengers on radiation-induced DNA double strand breaks.

PURPOSE: To investigate possible effects of Tris and phenol on the dynamic properties of gamma-irradiated DNA molecules in addition to their well known scavenging capacity. MATERIALS AND METHODS: Native and fragmented calf thymus DNA molecules were exposed to various doses of 60Co gamma-rays at approximately 4.5Gy/min. Using thermal transition spectrophotometry, pulsed field gel electrophoresis and standard agarose gel electrophoresis, the effects of Tris, phenol and NaCl on the double helix to single coil thermal transition temperature, Tm, and the yield of the double-strand breaks (Gdsb) of the irradiated DNA molecules have been studied. RESULTS: DNA molecules exposed to gamma-rays showed a decreased Tm and a corresponding increase of the Gdsb yield. Tris, as well as phenol, exhibited a strong protection against preventing these radiation-induced alterations. In addition, both substances strongly affected the thermal stability of the non-irradiated DNA samples. These results, compared with data obtained by NaCl and its effects on DNA thermostability and Gdsb, revealed that in the presence of both scavengers the observed dsb decrease was correlated to an increased molecular stability of DNA. CONCLUSIONS: This work suggests that the total protective effect of Tris and phenol against radiation-induced dsb is mainly attributed to their well-known radical scavenging properties, while relatively minor protective effects arise from their contribution to an increased molecular stability of DNA.

Alpha-particle-induced changes in the stability and size of DNA.

The effect of alpha-particle radiation on the thermal stability and size of calf thymus DNA molecules in deoxygenated aqueous solutions was investigated by thermal transition spectrophotometry, pulsed-field gel electrophoresis, and standard agarose gel electrophoresis. The thermal transition of DNA from helix to coil was studied through analysis of the UV A(260) absorbance. The results obtained for alpha particles of mean LET of 128 keV microm(-1) reveal a dual dose response: a tendency for thermal stability of the DNA helix at "low" doses, followed by an increasing instability at higher doses. The same phenomenon was observed for the mean molecular weight of DNA molecules exposed to alpha particles. The results reported here for alpha particles in the low-dose region of 0-16 Gy are consistent with our previous hypothesis of inter- and intramolecular interactions of a covalent character in gamma-irradiated DNA molecules in the dose region of 0-4 Gy.

Low doses of alpha- and gamma-radiation enhance DNA thermal stability.

Isolated calf thymus DNA in buffered solutions has been exposed to 0-150 Gy of alpha- and gamma-radiation. The effects of alpha- and gamma-radiation on the thermal stability and electrophoretic mobility of the DNA molecules have been studied by UV spectroscopic 'melting' and Pulsed Field Gel Electrophoresis (PFGE), respectively. The observed thermal denaturation parameters were fitted to the energy propagation descriptive model. The experimental results for the samples exposed to relatively low (low) doses indicate an increased thermal stability and a reduced mobility over that of the controls. The expected overall degradation of the DNA molecules was confirmed for the samples exposed to high doses. Our results are in good agreement with the predictions of the energy propagation model, which now is also tested in the low dose region and for an additional type of ionising radiation (alpha-particles). Our findings are consistent with conformational changes at low doses resulting in a DNA form characterised by localised alterations, which affect the energy flow along the DNA molecule.

Effects of gamma rays on the stability and size of DNA.

The effects of gamma radiation on the stability and size of mammalian DNA were studied by using thermal transition spectrophotometry and pulsed-field and standard agarose gel electrophoresis. The experiments were performed using deproteinized calf thymus DNA in buffered deaerated aqueous solutions. A dual dose response was observed: a tendency for increased helix stability at "low" doses (0-4 Gy) accompanied by a high tendency of the DNA molecules to interact, forming larger molecules, followed by a gradual increase of degradation and helix instability at higher doses. The results reported here for the low-dose region are consistent with the hypothesis of inter- and intramolecular interactions of covalent character (crosslinking) in irradiated DNA molecules.

[Sensitized NADH formation of single-stranded breaks in plasmid DNA upon the action of near UV-radiation]. / Sensibilizirovannoe NADH obrazovanie odnonitevykh razryvov v plazmidnoi DNK pri deistvii blizhnego UF-izlucheniia.

It has been shown that NADH photosensitize in vitro single-strand breaks formation in double-strand plasmid DNA pBR 322 upon near-UV (320-400 nm) irradiation. The number of single-strand breaks depends both on UV light dose and sensitizer concentration. Addition of catalase and sodium benzoate strongly decreases the single-strand breaks formation. The results show an important role of hydrogen peroxide (H2O2) and hydroxyl radical (.OH) in inducing single-strand breaks in plasmid DNA irradiated by near-UV radiation in the presence of NADH.

Mutagenesis; carcinogenesis and the metal elements--DNA interaction.

The present work presents evidence indicating that copper, which binds on the phosphate and base moieties of the DNA, induces single strand DNA breaks and increases the frequency of mutations and sister chromatid exchanges in cultured V-79 fibroblast cells, while in solution decreases the thermostability of the DNA of these cells. Magnesium which binds only at the phosphate moiety does not induce lesions in the genetic material of the V-79 cells. A more general hypothesis is proposed relating to mutagenic/carcinogenic potential of metal elements to their affinity for binding on the base moiety of DNA.

Biological effectiveness of low energy protons. I. Survival of Chinese hamster cells.

The biological effectiveness of monoenergetic protons was investigated with the track-segment method. Protons were accelerated by a Tandem Van de Graaff accelerator and their final energies were 3.0 and 7.4 MeV. The biological system used was Chinese hamster V-79 cells and their survival ability following proton irradiation was investigated. Cobalt-60 gamma-rays were used as reference radiation to assess proton relative biological effectiveness (RBE). Survival curves were obtained for the gamma-ray and proton irradiations, and the relation S = exp (-alpha D-beta D2) was fitted to the data and the parameters alpha and beta were determined. The RBE values, calculated on the basis of the mean inactivation dose D and other pertinent parameters, were found to be 1.7 +/- 0.1 and 2.8 +/- 0.2 for 7.4 and 3.0 MeV protons, respectively. Comparisons were made with the results published by other investigators and it was concluded that in this low energy range the biological effectiveness increases substantially with decreasing proton energy.

Perturbed gamma-gamma angular correlation studies of 111In bound to double and single stranded DNA.

Using 111In as a probe, the dynamics of double and single stranded DNA have been studied by performing time-differential gamma-gamma angular correlation measurements on the 173-247 keV transitions in 111Cd. Binding of indium to DNA was investigated by analysis of sedimentation profiles, thermal transition spectrophotometry and electrochromatography. Rotational correlation times tau c greater than or equal to 400 ns and tau c = 27 +/- 7 ns were obtained respectively for double stranded (native) and single stranded (denatured) DNA. The corresponding quadrupole interaction frequencies were omega 0 = 711 +/- 15 Mrad/s and omega 0 = 151 +/- 13 Mrad/s.

A simplified procedure for the observation in situ of chromosome aberrations or sister chromatid exchanges and the estimation of the mitotic index in mammalian monolayer cell cultures.

Chinese hamster V-79 cells are widely used in short term screening for potential physical or chemical mutagens of the environment. A simplified version of the standard Giemsa protocol of Moorhead and the Feulgen plus Giemsa protocol of Wolff and Perry is given which permits the observations in situ of chromosome aberrations or sister chromatid exchanges and the estimation of the mitotic index in the Petri dishes for the culture of the V-79 cells.

A spectrum response study on single strand DNA breaks, sister chromatid exchanges, and lethality induced by phototherapy lights.

Little information is available on the effect of visible light from commercial fluorescence lamps, commonly used in the treatment of neonatal hyperbilirubinemia, on parameters related to genetic damage in eucaryotic cells. The present study was undertaken to determine whether or not visible light of different wavelengths had any differential effects on the frequency of DNA breaks (frank breaks plus alkaline labile lesions), DNA replication, frequency of sister chromatid exchanges, and survival in cultured Chinese hamster cells. The results revealed that the "blue" spectral band (420 to 500 nm) is mainly responsible for DNA breaks, sister chromatid exchanges, and lethality induced by fluorescent light. This band is precisely that which bilirubin heavily absorbs and, hence, is the most efficient for the decomposition of this metabolite. These results were obtained with the use of light doses of 5 to 30 X 10(4) J/m3 versus the light doses of the order of 100 X 10(4) J/m2 being received by infants undergoing phototherapy treatments.

Decreased repair of x-ray induced DNA single-strand breaks in lymphocytes in Down's syndrome.

Gamma-ray irradiation introduces single and/or double strand breaks into the DNA molecule of the cells. In the case of mammalian cells, these breaks are being repaired in general during the first hr following exposure to ionizing radiation. The article reports on the results obtained from testing the ability of cultured lymphocytes from patients with Down's syndrome to repair radiation-induced DNA single-strand breaks. The ability to repair was deduced from the study of the DNA sedimentation profiles in alkaline sucrose gradients. It was found that lymphocytes from Down's syndrome patients are less efficient in repairing single-strand DNA breaks than are lymphocytes from normal individuals. This significantly increased fraction of unrepaired DNA strand breaks might be associated with the unusually high level of radiation-induced chromosome aberrations as compared with normals.