Characteristics of chromatin breakdown in the rat thymus after exposure to fast neutrons and gamma rays

A comparative study was made of the radiobiological aftereffects of the action of fast neutrons and gamma-rays on lymphoid tissues of rat thymus with a reference to a biochemical criterion of the interphase death of lymphocytes, i.e. the formation of polydeoxynucleotides (PDN). It was shown that the increase in the chromatin degradation was a function of dose of neutron- and gamma-radiation (up to 4 Gy). The dynamics of the PDN formation was similar with both types of radiation, but 4-6 h after neutron irradiation chromatin degradation was higher more pronounced. The RBE of neutrons varied from 3 to 2 with a radiation dose varying from 0.25 to 4 Gy.     

Acquired radioresistance of progeny of irradiated cells is accompanied by rearrangements in chromatin organization

gamma-Irradiation action within a dose range of 0-20 Gy on parental djungarian hamster fiborblasts, DH-TK- cell line, and the progenies of these irradiated cells, surviving acute exposure to 20 Gy irradiation, PIC-20 cell line, was examined. The PICs were 3 times more radioresistant than the parental cells as calculated from D0. Using a method of anomalous viscosity time dependence (AVTD) it was revealed that starting (initial) level (in untreated cells) of chromatin compactness in radioresistant progenies was more than 1.4 times as high as for parental cells. The analysis of dose dependence has shown that irradiation with a dose of 5 Gy resulted in complete chromatin loop relaxation in radiosensitive DH-TK- cells and partial one in radioresistant PIC-20 cells. Besides, the beginning of DNA-membrane complexes degradation following the irradiation with doses over 15 Gy in DH-TK- cells was observed. It was shown that the increased state of relative chromatin relaxation in PIC-20 cells determines an increasing in reparation effectiveness that resulted in lower percent of residual damages in these cells. Using the Nosern hybridization method the expression level of mts 1, tag 7 and vseap 1 genes was studied. It is revealed that tag 7 and vseap 1 gene expression in radioresistant cells were correspondingly 6 and 10 times higher than in radiosensitive parental cells and the level of mts 1 gene expression was not changed. So, based on the results obtained we suggest that acquired radioresistance in progenies of irradiated cells is determined by rearrangements in chromatin structure and accompanied constitutive changes of gene expression.     

A comparison of chromatin degradation in irradiated normal and tumorous lymphoid cells

Irradiation of mice with doses of 2 and 4 Gy induced extensive chromatin degradation in the thymocytes within 6 hours accompanied by an increase in polydeoxynucleotide (PDN) content (36 and 42 times, respectively). Fifteen hours after irradiation the PDN level was considerably lower, however, still being 4.7 and 14 times the control values after doses of 2 and 4 Gy. The PDN content in control LS/BL lymphosarcoma cells was similar as that in the thymocytes of non-irradiated mice. Unlike in the thymocytes, irradiation of lymphosarcoma cells did induce no statistically significant increase in the PDN level 6 and 15 hours after the irradiation, respectively. It has been reported previously (Matyásová et al. 1973) that chromatin of LS/BL cells degraded similarly as that in the irradiated thymocytes. The results of the present experiments thus provide additional evidence for changes of LS/BL cell properties due to long term cultivation. These cells, however, are still able to react by chromatin fragmentation to nitrogen mustard treatment.     

Mechanism of thymocyte death in exposure to ultrahigh doses of gamma radiation

A comparative study was made of the morphological and biochemical indices of rat thymus cells after gamma-irradiation with doses of 4-10 Gy (median), 20 Gy (high), and 200-400 Gy (superhigh). It was shown that 4 h after irradiation with superhigh doses the yield of polydeoxynucleotides (PDN) was twice as low as that observed after doses of 4-10 Gy. 24 h after irradiation the amount of the extracted PDN in thymocytes exposed to superhigh doses was markedly larger than that after 4 hours. After all doses applied chromatin degradation occurred at the internucleosome sites in a strict order, the activity of acid and alkaline nucleases being unchanged. A large number of cells have normal nuclear structure 4 h after irradiation (200-400 Gy), as was demonstrated by the electron microscopy data, while in 24 h no intact cells were virtually found in the thymus which correlated with the changes in the PDN yield. The mechanisms of the lymphoid cell death under the effect of different radiation doses are discussed.     

Electrophoretic analysis of the internucleosome fragmentation of DNA in irradiated thymocytes of rats

Total DNA and DNA of chromatin degradation products obtained from rat thymocytes 6 h after irradiation with a dose of 10 Gy were separated electrophoretically. Relative shares of mononucleosomes and their oligomers were determined. Experimental distributions of DNA fragments differ from those calculated on the basis of the assumption of a random breakage of bonds between the nucleosomes.     

Irradiation damage in chromatin isolated from V-79 Chinese hamster lung fibroblasts

The effect of chromatin structure on the extent of radiation damage induced by low doses of 100 KeV X rays was investigated using a fluorescent assay for DNA unwinding. Chromatin was isolated from V-79 Chinese hamster lung fibroblast nuclei by partial digestion with micrococcal nuclease. Gel electrophoresis of the isolated DNA showed the molecular weight of the chromatin preparation to be 10.6 X 10(6) with a size range of 6.6-21.7 X 10(6) Da while a size of 10.2 +/- 0.9 X 10(6) Da was found by sedimenting the DNA in alkaline sucrose gradients. The repeat length of V-79 chromatin was found to be 194 +/- 3 bp. The typical nucleosomal repeat structure of the isolated chromatin and that of intact nuclei was identical. Irradiation with 50 and 100 Gy of 100 KeV X rays and analysis by alkaline sucrose density centrifugation indicated that V-79 chromatin sustained 0.56 +/- 0.19 and 0.69 +/- 0.09 single-strand breaks per 10 Gy per 10(8) Da of DNA, respectively. Irradiation with doses of 0.5-3.0 Gy of 100 KeV X rays and analysis by the fluorometric assay showed that the radiation sensitivity of V-79 chromatin decreases sharply on compaction with MgCl2. Histone H1 depletion, which inhibits compaction and causes chromatin to expand by increasing the linker from 26 to 48 bp, results in a considerable increase in the radiation sensitivity. It is concluded that radiation damage sustained by DNA is greatly influenced by chromatin structure.     

DNA Base Damage in Chromatin of γ-Irradiated Cultured Human Cells

We report on the chemical characterization of DNA base damage in chromatin of γ-irradiated cultured human cells. Chromatin was isolated from unirradiated and irradiated cells and analyzed by gas chroma-tography/mass spectrometry with selected-ion monitoring after acidic hydrolysis of chromatin and trimethylsilylation of hydrolysates. Prior to analysis of chromatin samples, experimental conditions for acidic hydrolysis were optimized by determining the relative molar response factors of modified bases under non-acidic and acidic conditions, and their release from DNA under various acidic conditions. A number of modified bases in chromatin isolated from irradiated cells were identified and quantitated. These were 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, 5-(hydroxymethyl)uracil, cytosine glycol, thymine glycol, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine. Radiation doses ranging from 42 to 420 Gy (J . kg¹) were used. Background levels of all modified bases were observed in chromatin isolated from unirradiated cells. The radiation yields of a number of modified bases were increased significantly over their background levels at a dose as low as 42 Gy. In most cases, linear dose-yield relationships were obtained up to ≈200Gy. At radiation doses higher than 420 Gy, no additional increase in the yields of modified bases was observed. The yields of guanine-derived bases amounted to ≈ 45% of the total net yield of modified bases measured, followed by almost equal yields of adenine-, cytosine- and thymine-derived bases. Modified bases identified were typical products of hydroxyl radical attack on DNA bases, indicating the involvement of hydroxyl radical, although their induction in part by the direct effect of ionizing radiation through ionization of DNA bases cannot be excluded. The yields of modified bases were lower than those previously measured after γ-irradiation of fully expanded chromatin in aqueous buffer solutions.     

Structural rearrangements of the chromatin during the adaptive response in two genetically close djungarian hamster fibroblast cell lines with different radiosensitivity

The capacity of cell for the adaptive response (AR) induction after gamma-irradiation using micronuclear test was investigated. Our model consists of the parental djungarian hamster embryonic fibroblast cell line DH-TK- and its radioresistant progeny (PIC-20). We demonstrated that AR for the more radiosensitive parental cell line was shifted to the lower adaptive and to the challenge doses. The maximal AR for DH-TK- cells was induced at 0.3 Gy adaptive dose and 1.5 Gy challenge dose (adaptive response coefficient (ARC) was 0.4+/- 0.1), whereas for PIC-20 cells these means were 0.5 Gy and 3.0 Gy correspondingly (ARC = 0.45+/-0.1). Using the method of anomalous viscosity time dependence (AVTD) we demonstrated the chromatin rearrangements in both cell lines during 3-5 h after adaptive dose application. The rearrangement degree evaluated by the relative maximal reduced viscosity was considerably higher in PIC-20 cell line than that in DH-TK cells (2.4+/-0.3 vs 1.4+/-0. 1). Interestingly, the time of chromatin rearrangement did not depend neither on the dose nor on the cell type and was similar in both cell lines after 5 h of adaptive dose application. It was also shown that during the AR chromatin relaxation was lower after exposure to both the adaptive and challenge doses than after challenge dose only. In contrast, in the degree of AR chromatin relaxation was higher for both cell lines.     

Phospholipids in the nuclei and chromatin of the rat thymus at long periods following gamma-irradiation

The phospholipid content of the homogenate, nuclei and chromatin of rat thymus was being studied during three months after fractionated gamma-irradiation (2 Gy X 3 at a week interval). The number of phospholipids in the total fraction of phosphatidyl choline + phosphatidyl serine and phosphatidyl ethanolamine in the nuclei and chromatin of rat thymus was shown to decrease 60 min following the last exposure. In a month the phospholipid content in the nuclei and chromatin increased up to the control level keeping it throughout the entire period of observation.     

Changes in chromatin conformation during radiation-induced apoptosis in human lymphocytes

Human peripheral lymphocytes in G(0) phase were irradiated with 1-5 Gy of gamma rays. The biochemical and morphological changes characteristic of apoptosis were examined for 72 h after irradiation. In parallel, changes in chromatin conformation were studied by the method of anomalous viscosity time dependence (AVTD) and by measurements of nuclear halo size. An immediate and dose-dependent relaxation of chromatin, which became saturated at doses above 2-3 Gy, was revealed by the AVTD method. The state of relaxed chromatin lasted up to 12-24 h after irradiation, a response considerably longer than the time attributable to repair of radiation-induced DNA breaks. Measurements of nuclear halo size also indicated the initial relaxation of chromatin in the irradiated cells and its subsequent condensation. This condensation of chromatin as revealed with AVTD correlated well with nuclear condensation, as measured with dual fluorescence staining, and with DNA fragmentation, as measured by conventional and pulsed-field gel electrophoresis (PFGE). Late apoptotic cells did not contribute significantly to the AVTD signal, showing that the chromatin of these cells was completely condensed and fragmented.     

Methylation and degradation of chromatin DNA in isolated rat liver cell nuclei]

Methylation of chromatin DNA in rat liver cell nuclei incubated in a medium with [3H]CH3-S-adenosyl methionine was studied. It was shown that under the given experimental conditions DNA methylation and chromatin degradation by endogenous nuclear nuclease (nucleases) with a formation of chromatin structural subunits occur simultaneously. An analysis of methylated chromatin DNA degradation products based on a number of approaches demonstrated a predominant methylation of extra-nucleosomal DNA. The data obtained suggest that chromatin of isolated nuclei contain sites with supermethylated DNA fragments incorporating not less than 400 nucleotide pairs. These sites possess an increased sensitivity to endogenous nuclease.     

Effects of ubiquinone-9 on lipids of nuclei and chromatin of the rat liver under continuous irradiation]

The influence of continuous gamma irradiation on the lipids of nuclei and chromatin of rat liver at a dose-rate of 0,129 Gy/day for 155 days (a total dose of 20 Gy) and by feeding of ubiquinone-9 has been studied. The amount of phosphatidylcholine with phosphatidylserine and phosphatidyl-ethanolamine in liver nuclei of irradiated rats was found to increase. Ubiquinone-9 had a normalizing effect. A decrease of cardiolipin was observed in the liver chromatin of irradiated rats. The amount of free fatty acids had a tendency to decrease in homogenate, nuclei and liver chromatin of irradiated rats. Ubiquinone was found to increase the amount of free fatty acids up to the control level. The amount of cholesterol in nuclei was increased after irradiation and that in chromatin tended to rise. Ubiquinone-9 significantly decreased the amount of cholesterol in nuclei and chromatin of irradiated rats.     

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.     

Investigation of the pollen chromatin state of Hippeastrum hibridum at the early stages of development with fluorescent dye Hoechst 33342

The work was carried out on the pollen of Hippeastrum hibridum, whose size (ca 55 mcm) permits to follow both the pollen tube formation and development of the vegetative nucleus and generative cell within the first two hours before the growing point arising. Using fluorescent dye Hoechst 33342 chromatin state alterations accompanying changes in the pollen physiological state were investigated. The maximum fluorescent intensity was observed in 30 min of staining and reflected the maximum chromatin functional activity. Histograms of pollen distribution according to fluorescent intensity differ considerably with doses of irradiation (500, 1000, 2000, 3000 and 4000 Gy). Besides, a germinative index of fluorescence intensity was calculated. A comparative analysis of these data has shown that the lowest decrease in fluorescent intensity observed at 500, 3000 and 4000 Gy, was accompanied by different germinative indices. At 500 Gy, the index was 2.5 times lower than in the control, but at 4000 G no germination was not noted. The process of pollen grain development is supposed to be more intensive and faster at 500 Gy, than in the control. At 4000 Gy, a decrease in the functional pollen activity is accompanied by decrease or inhibition of chromatin functional activity.     

Autoradiographically detected impairment of RNA synthesis pattern in 8-16 cell bovine embryos after irradiation

Early preimplantation bovine embryos at 8- or 16-cell stage were analysed by [5-3H]uridine autoradiography for distribution of newly synthesized RNA after 60Co irradiation with a single dose of 1 Gy, 2 Gy or 4 Gy gamma rays, respectively. Embryos irradiated with a single dose of 1 Gy showed equally decreased synthesis of RNA in nucleoplasma as well as in nucleolus. In embryos irradiated with a single dose of 2 Gy or 4 Gy, RNA synthesis was decreased and localized mostly on the periphery of the nucleus; in both cases of irradiation, the nucleus center being without labelling. In most of embryos irradiated with a dose of 4 Gy, the nucleoli were not labelled, and an increasing occurrence appeared of various nucleus chromatin segregation forms, mainly as its marginalization.     

Dissociation and reconstitution of chromatin without appreciable degradation of the proteins.

When chromatin from Novikoff hepatoma ascites cells was dissociated in 3 M NaCl – 7 M urea either at pH 6 or 8, degradation of chromosomal proteins was observed in two-dimensional gel electrophoretic patterns. This degradation was not prevented by 50 mM NaHSO₃ but was prevented by 1 mM PMSF (phenylmethylsulfonyl fluoride). Reconstitution of the chromatin components dissociated in 3 M NaCl – 7 M ure ? 0.05 M sodium acetate (pH 6.0) containing 1 mM PMSF resulted in reassociation of DNA, histones and the major nonhistone proteins (B24, B26, B33, BE, BJ, C1, C6, CG, CH, CM, C14, CP, C18, CR, CS and C25). Two-dimensional gel electrophoresis showed that although the proportion of the nonhistone proteins to histones was lower in reconstituted than in native chromatin, the template activity of the reconstituted chromatin was similar to that of native chromatin.     

The effect of low doses of external gamma irradiation on the chromatin structure and activity of the histone-specific proteinases of rat brain cells]

Irradiation of rats with doses of 0.5 to 2 Gy was shown to cause dose-dependent changes in the sensitivity of brain cell chromatin to the effect of DNAase I that were manifested by the increased level of DNA hydrolysis and a high content of the chromatin soluble fractions. The chromatin structure was only partially restored 24 h after irradiation. Changes in the chromatin structure were accompanied by the increase in the histone-specific proteinase activity.     

Prediction of radiosensitivity in human bladder cell lines using nuclear chromatin phenotype.

BACKGROUND: Nuclear texture analysis measures phenotypic changes in chromatin distribution within a cell nucleus, while the alkaline Comet assay is a sensitive method for measuring the extent of DNA breakage in individual cells. The authors aim to use both methods to provide information about the sensitivity of cells to ionizing radiation. METHODS: The alkaline Comet assay was performed on six human bladder carcinoma cell lines and one human urothelial cell line exposed to gamma-radiation doses from 0 to 10 Gy. Nuclear chromatin texture analysis of 40 features was then performed in the same cell lines exposed to 0, 2, and 6 Gy to explore if nuclear phenotype was related to radiation sensitivity. RESULTS: Comet assay results demonstrated that the cell lines exhibited different levels of radiosensitivity and could be divided into a radiosensitive and a radioresistant group at >6 Gy. Using stepwise discriminant analysis, a subset of important nuclear texture features that best discriminated between sensitive and resistant cell lines were identified A classification function, defined using these features, correctly classified 81.75% of all cells into their radiosensitive or radioresistant groups based on their pretreatment chromatin phenotype. Posttreatment chromatin changes also varied between cell lines, with sensitive cell lines showing a relaxed chromatin conformation following radiation, whereas resistant cell lines exhibited chromatin condensation. CONCLUSIONS: The authors conclude that the alkaline Comet assay and nuclear texture methodologies may prove to be valuable aids in predicting the response of tumor cells to radiotherapy.     

Characteristics of the development of interphase death of hematopoietic tissue in dogs in response to gamma irradiation

In experiments with 2-3 week dogs it was shown that whole-body gamma-irradiation with a dose of 3 Gy causes an insignificant absolute rise in the amount of degrading chromatin of the haemopoietic organs during the first 24 h following irradiation. After 48 h, this cell death parameter is normalized. A considerably lower radiosensitivity of lymphoid cells of dogs compared to small laboratory animals is indicated by a stable DNA content per 1 g of thymus, intactness of its structure after irradiation, and the absence of an increase in thymidine content of blood of young and adult dogs under the effect of 3-3.7 Gy radiation.