Action of secondary radiation from 70-Gev protons on the cellular DNA of mammals

A study was made of the effect of secondary radiation of 70 GeV protons on DNA of Chinese hamster cells. With a reference to fibroblast DNA, lymphoid cell DNA, and the lethal effect of radiation on the survival of Chinese hamster cells the RBE was 1.6-7.6, 1.1-3.8 and 1.14-1.7, respectively. DNA breaks were repaired to an equal level after exposure to secondary radiation from the accelerator and gamma-radiation from 60Co in equally effective doses.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

Enzymatic detection of x-ray-induced strand breaks in nuclear DNA on sections of mouse tissue.

An enzyme-histochemical method was used to detect X-ray-induced strand breaks in the DNA of mammalian tissue cells. Paraffin sections of ethanol-fixed mouse brain and liver were incubated with three kinds of exogenous DNA polymerizing enzymes, and the amount of in situ incorporation of 3H-deoxyribonucleotides into nuclear DNA was examined by autoradiography. No increase in labelling intensity was observed over nuclei of neurons and astrocytes in cerebral cortex, or over hepatocytes in liver immediately after X-irradiation when compared with unirradiated specimens. In liver Kupffer cells, heavily-labelled nuclei appeared from 30 min to 6 hours after, but were not observed immediately after X-irradiation. This method cannot, therefore, be applied to detect the strand breaks directly induced by X-rays, but it is useful in detecting secondary DNA degradation occurring as a result of nuclear degradation.     

Detection of ionizing radiation-induced DNA double-strand breaks by filter elution is affected by nuclear chromatin structure

Chinese hamster ovary cells were irradiated with 250 kVp X rays and analyzed for the presence of DNA double-strand breaks using either polycarbonate filter elution or pulsed-field agarose gel electrophoresis at neutral pH. Reduction in DNA length detected by filter elution was produced as a nonlinear function of increasing radiation dose, with a quasi-threshold at low total dose, and as a first-order function of increasing radiation dose as detected by gel electrophoresis. The quasi-threshold observed with filter elution was eliminated when nuclei were isolated from irradiated cells and their chromatin relaxed in a buffer containing low-molarity monovalent cation prior to analysis by filter elution. The results suggest either that the chemical structure of the DNA double-strand breaks produced by low-LET radiation necessitates a DNA relaxation step before they can be detected accurately by filter elution, or that at low total radiation dose a DNA complex forms on the polycarbonate filter.     

Comparison of the protective effect of garlic extract and cell defense during adaptive response

The resistance of human cell DNA to damaging doses of CdCl2 or gamma radiation has been investigated after pretreatment with garlic extract (GE) or with adaptive doses of the same mutagens. The adaptive response (AR) and pretreatment with GE stabilize the DNA structure in a similar way. In experiments with 4-nitroquinoline-1-oxide (4-NQO), GE does not stabilize DNA structure but increases the rate and volume of repair of induced breaks. 3-Aminobenzamide (3-AB) increases the number of DNA breaks induced in experiments with CdCl2, gamma radiation, and 4-NQO. This suggests that poly(ADP-ribose)polymerase participates defense of cells from mutagens. Thus, it has been demonstrated that cell defense from CdCl2 or gamma radiation in experiments with GE and AO is mediated by stabilization of DNA structure and in experiments with 4-NQO, by activation of repair of DNA breaks induced.     

Locations of radiation-produced DNA double strand breaks along chromosomes: a stochastic cluster process formalism.

Ionizing radiation produces DNA double strand breaks (DSBs) in chromosomes. For densely ionizing radiation, the DSBs are not spaced randomly along a chromosome: recent data for size distributions of DNA fragments indicate break clustering on kbp-Mbp scales. Different DSB clusters on a chromosome are typically made by different, statistically independent, stochastically structured radiation tracks, and the average number of tracks involved can be small. We therefore model DSB positions along a chromosome as a stationary Poisson cluster process, i.e. a stochastic process consisting of secondary point processes whose locations are determined by a primary point process that is Poisson. Each secondary process represents a break cluster, typically consisting of 1-10 DSBs in a comparatively localized stochastic pattern determined by chromatin geometry and radiation track structure. Using this Poisson cluster process model, which we call the randomly located clusters (RLC) formalism, theorems are derived for how the DNA fragment-size distribution depends on radiation dose. The RLC dose-response relations become non-linear when the dose becomes so high that DSB clusters from different tracks overlap or adjoin closely. The RLC formalism generalizes previous models, fits current data adequately and facilitates mechanistically based extrapolations from high-dose experiments to the much lower doses of interest for most applications.     

Asymmetric somatic cell hybridization in plants

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.     

A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells

A PFGE method was adapted to measure DNA double-strand breaks (DSBs) in mammalian cells after low (0-25 Gy) doses of ionising radiation. Instead of radionuclide incorporation, DNA staining in the gel by SYBR-Gold was used, which lowered the background of DNA damage and could be applied to non-cycling cells. DSB level was defined as a product of a fraction of DNA released to the gel (FR) and a number of DNA fragments in the gel (DNA(fragm)) and expressed as a percentage above control value. The slope of the dose-response curve was two-fold higher compared to that with FR alone as DSB level indicator (31.4 versus 15.6% per Gy). Two alternative ways were proposed to determine the total amount of DNA, used for FR calculation: measurement of DNA content in a plug not subjected to electrophoresis, with the use of Pico-Green, or estimation of DNA released to the gel from a plug irradiated with 600 Gy of gamma-rays. The limit of DSB detection was 0.25 Gy for human G1-lymphocytes and 0.5-1 Gy for asynchronous cultures of human glioma M059 K and J or mouse lymphoma L5178Y-R and -S cells. Specificity of our PFGE assay to DSB was confirmed by the fact that no damage was detected after treatment of the cells with H(2)O(2), an inducer of single-strand DNA breaks (SSBs). On the contrary, the H(2)O(2) inflicted damage was detected by neutral comet assay, attaining 160% above control (equivalent to 2.5 Gy of X-radiation). DSB rejoining, measured in cells after X-irradiation with a dose of 10 Gy, generally proceeded faster than that measured previously after higher (30-50 Gy) doses of ionising radiation. Clearly seen were defects in DSB rejoining in radiosensitive M059 J and L5178Y-S cells compared to their radioresistant counterparts, M059 K and L5178Y-R. In some cell lines, a secondary post-irradiation increase in DSB levels was observed. The possibility is considered that these additional DSBs may accumulate during processing of non-DSB clustered DNA damage or/and represent early apoptotic events.     

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of ¹³⁷Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after the incubation of 2'-deoxyguanosine (dGuo) with ChOOH and Cu(2+). In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

The DNA ligase III zinc finger stimulates binding to DNA secondary structure and promotes end joining

The ability to rejoin broken chromosomes is fundamental to the maintenance of genetic integrity. Mammalian cells possess at least five DNA ligases, including three isoforms of DNA ligase III (Lig-3). Lig-3 proteins differ from other DNA ligases in the presence of an N-terminal zinc finger (Zn-f) motif that exhibits extensive homology with two zinc fingers in poly(ADP-ribose) polymerase (PARP). Here we report that the Zn-f confers upon Lig-3 the ability to bind DNA duplexes harbouring a variety of DNA secondary structures, including single-strand gaps and single-strand flaps. Moreover, the Zn-f stimulates intermolecular end joining of duplexes that harbour these structures up to 16-fold. The Zn-f also stimulates end joining between duplexes lacking secondary structure, but to a lesser extent (up to 4-fold). We conclude that the Zn-f may enable Lig-3 to rejoin chromosomal DNA strand breaks located at sites of clustered damage induced by ionising radiation or near to secondary structure intermediates of DNA metabolism.     

Protection against genotoxic damages following whole body gamma radiation exposure in mice by lipoic acid

Alpha-lipoic acid (LA) protected plasmid pBR 322 DNA, under in vitro conditions from gamma radiation induced strand breaks as evidenced by the prevention of the loss of supercoiled covalently closed circular form upon irradiation. It also protected the membrane lipids of liver homogenates from the oxidative damages. Whole body exposure of mice to gamma-radiation resulted in damage to cellular DNA in various tissues and administration of LA prior to the radiation exposure prevented the radiation induced DNA damage as assessed by alkaline comet assay. Administration of LA to mice prior to the radiation exposure also prevented induction of chromosomal damages in bone marrow cells and formation of micronuclei in blood reticulocytes. Thus taken together, LA a normal cellular constituent could be used as a radioprotector against whole body radiation exposure scenarios.     

激光对DNA作用机理的AFM研究

激光作用质粒DNA和小牛胸腺DNA产生损伤效应,导致DNA结构变化,利用一种改进的试样制备过程和纳米显微镜－－原子力显微镜（AFM）能够获得可重现的激光作用质粒DNA和小牛胸腺DNA的AFM图像,显示它们的特殊的表达结构,讨论了激光辐照导致DNA链断裂的作用机理。     

Rejoining kinetics of DNA single- and double-strand breaks in normal and DNA ligase-deficient cells after exposure to ultraviolet C and gamma radiation: an evaluation of ligating activities involved in different DNA repair processes

The repair kinetics for rejoining of DNA single- and double-strand breaks after exposure to UVC or gamma radiation was measured in cells with deficiencies in DNA ligase activities and in their normal counterparts. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III activity as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. DNA single- and double-strand breaks were assessed by the comet assay in alkaline conditions and by the technique of graded-field gel electrophoresis in neutral conditions, respectively. 46BR cells, which are known to re-ligate at a reduced rate the DNA single-strand breaks incurred during processing of damage induced by UVC but not gamma radiation, were shown to have a normal repair of radiation-induced DNA double-strand breaks. EM9 cells exhibited a reduced rate of rejoining of DNA single-strand breaks after exposure to ionizing radiation, as reported previously, as well as UVC radiation. EM-C11 cells were deficient in the repair of radiation-induced-DNA single-strand breaks but, in contrast to EM9 cells, demonstrated the same kinetics as the parental cell line in the resealing of DNA breaks resulting from exposure to UVC radiation. Both EM9 and EM-C11 cells displayed a significant defect in rejoining of radiation-induced-DNA double-strand breaks. XR-1 cells were confirmed to be highly deficient in the repair of radiation-induced DNA double-strand breaks but appeared to rejoin DNA single-strand breaks after UVC and gamma irradiation at rates close to normal. Taken together these results indicate that: (1) DNA ligase I is involved only in nucleotide excision repair; (2) DNA ligase IV plays an important role only in repair of DNA double-strand breaks; and (3) DNA ligase III is implicated in base excision repair and in repair of DNA double-strand breaks, but probably not in nucleotide excision repair.     

Chromatin organization contributes to non-randomly distributed double-strand breaks after exposure to high-LET radiation

The influence of higher-order chromatin structure on the non-random distribution of DNA double-strand breaks induced by high-LET radiation was investigated. Five different chromatin structures (intact cells, condensed and decondensed chromatin, nucleoids and naked genomic DNA) from GM5758 cells or K562 cells were irradiated with (137)Cs gamma-ray photons and 125 keV/microm nitrogen ions (16-25 MeV/nucleon). DNA was purified with a modified lysis procedure to avoid release of heat-labile sites, and fragment size distributions and double-strand break yields were analyzed by different pulsed-field gel electrophoresis protocols. Whereas double-strand breaks in photon-irradiated cells were randomly distributed, irradiation of intact K562 cells with high-LET nitrogen ions produced an excess of non-randomly distributed DNA fragments 10 kb-1 Mbp in size. Complete removal of proteins eliminated this non-random component. There was a gradual increase in the yield of double-strand breaks for each chromatin decondensation step, and compared to intact cells, the yields for naked DNA (in buffer without scavengers) increased 83 and 25 times after photon and nitrogen-ion irradiation, respectively. The corresponding relative biological effectiveness decreased from 1.6-1.8 for intact cells to 0.49 for the naked DNA. We conclude that the organization of DNA into chromatin fiber and higher-order structures is responsible for the majority of non-randomly distributed double-strand breaks induced by high-LET radiation. However, our data suggest a complex interaction between track structure and chromatin organization over several levels.     

DNA complex lesions induced by protons and ⋅-particles: track structure characteristics determining linear energy transfer and particle type dependence

The yield of DNA double-strand breaks (dsb) and DNA complex lesions induced by protons and α-particles of various energies was simulated using a Monte Carlo track structure code (MOCA15) and a simple model of the DNA molecule. DNA breaks of different complexity were analysed. The linear energy transfer (LET) and particle-type dependence of lesions of higher complexity seems to confirm the importance of clustered damage in DNA as a relevant step leading to biological endpoints such as cell inactivation. The detailed structure of proton and α-particle tracks was analysed to identify the main characteristics possibly responsible for such a dependence. The role of the primary ion and of its secondary electrons in inducing dsb and complex lesions is described, showing that the relative contribution of secondary electron tracks alone in inducing clustered lesions is almost negligible at high LET, but tends to dominate below ≈10 keV/μm. This is consistent with the observed similar effectiveness of low-LET fast particle radiation and sparsely ionizing radiation such as x-rays. The dependence on LET and particle type is mainly due to energy deposition events of the primary ion together with short range electrons surrounding the ion track; the yield of complex lesions due to secondary electron tracks alone is substantially LET independent. The radial distributions of the energy contributing to the induction of complex lesions were analyzed and compared with the radial distributions of energy deposition of the full tracks. The results suggest that the stochastic behaviour (i.e. cluster properties) of the energy deposition pattern within a radius of a few nanometers around the ion track plays a relevant role in determining the biological radiation effectiveness. Received: 20 December 1996 / Accepted in revised form: 5 March 1997     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Abstract

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) after the incubation of 2′-deoxyguanosine (dGuo) with ChOOH and Cu²⁺. In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

Protection of DNA from gamma-radiation induced strand breaks by Epicatechin

Epicatechin (EC), a polyphenolic antioxidant compound found in tea, apples and chocolate offered protection to DNA against ionizing radiation induced damages. Under in vitro conditions of radiation exposure, plasmid pBR322 DNA was protected by EC in a concentration dependent manner. The dose modifying factor for 0.2 mM EC for 50% protection of the plasmid DNA was found to be 6.0. EC when administered to mice 1 h prior to exposure to 4 Gy gamma-radiation protected cellular DNA against radiation-induced strand breaks in peripheral blood leukocytes, as revealed in alkaline comet assay studies. Thus, EC was found to protect DNA from gamma-radiation indiced strand breaks under in vitro as well as in vivo conditions of radiation exposure.     

Stability of lipid oxidation products in the rat liver and pathways of their utilization

The ability of liver homogenates to utilize various lipid peroxidation products was studied. Conjugated dienes and TBA-reactive products of unsaturated fatty acid phospholipids and triglycerides were found to be more stable that the corresponding lipid hydroperoxides. It was shown that decomposition of lipid hydroperoxides in liver homogenates is due to their reduction to corresponding oxycompounds without activation of free radical reactions. The ability of lipid hydroperoxides to be reduced in liver homogenates is determined by their chemical structure and decreases in the following order: polyunsaturated fatty acids--phospholipids--triglycerides--cholesterol esters.