Change in survival of cells in the first 2 generations after irradiation and exposure to inhibitors of repair synthesis

In experiments on asynchronous population of HeLa S3 cells a study was made of the possibility of assessing DNA lesions which remained unrepaired for a long period of time following gamma-irradiation: in generation "O" directly affected by radiation and in generation "I" following the irradiated one. The presence of DNA damages was estimated by the reduction in survival of exposed cells incubated with inhibitors of repair and replicative syntheses of DNA, namely, with arabinoside cytosine and hydroxyurea. A considerable enhancement of the radiation effect was noted with the inhibitors added 0-6 h after irradiation (generation "O"), and a marked increase in the cell death was registered with the preparations injected 24-30 h after exposure (generation "I"). It is assumed that minor residual lesions persist in the generation of cells, following the one directly affected by gamma-radiation, which have completed the first postirradiation mitosis.     

Effects of inhibitors on repair of DNA in normal human and xeroderma pigmentosum cells after exposure to x-rays and ultraviolet irradiation

Experiments were carried out to obtain direct evidence for the hypothesis that in human cells the repair of UV-damaged DNA is initiated by an incision step, and that this step is defective in cells from patients having Xeroderma pigmentosum (XP). The alkaline sucrose gradient centrifugation technique was used to detect breaks in the DNA.A decreased sedimentation velocity of the DNA was found after exposure of normal and XP cells to high doses of UV (5000 erg/mm²). Breaks were induced in the DNA by the UV irradiation without the action of an enzyme. After exposure of both types of cell to UV doses of 100–500 erg/mm², breaks that might occur by enzymic incision were not observed, possibly because of immediate rejoining.After single-strand breaks had been induced by X-rays, rejoining did not occur at temperatures lower than 22°. Rejoining was inhibited by KCN, 2,4-dinitrophenol, EDTA, iodoacetate and crystal violet. Actinomycin D, acriflavine and phleomycin, also tested as potential inhibitors of the repair process, induced breaks or conformational changes in the DNA of unirradiated normal and XP cells.Application to UV-exposed cells of conditions that inhibit the rejoining of breaks did not cause accumulation of breaks in the DNA. The results suggest a coordinated and sequential performance of the steps in the repair of each UV lesion by repair enzymes which may act as a complex.     

Retention of tumorigenicity in radioresistant progeny of cells surviving exposure to high doses of gamma irradiation

The cell tumorigenic ability and the cell clonogenicity in semi-solid medium of highly radioresistant variant cell line, PIC-20 (the progeny of djungarian hamster fibroblast cell line DX-TK- surviving acute exposure to 20 Gy of gamma-irradiation), were examined. In the absence of additional radiation, no differences between tested features of non-irradiated PIC-20 cells and parental DX-TK- cells were observed. On the contrary, after gamma-irradiation with high doses the essential differences in the properties of the examined cell lines were revealed. After exposure to 10 Gy the surviving fraction of PIC-20 cells was 20 times higher than that of the parental cells. Both irradiated and non-irradiated PIC-20 cells produced colonies of similar size. It is revealed that even after irradiation with doses of 5, 10 or 15 Gy, the PIC-20 cells kept their tumorigenicity as high as non-irradiated ones. In all these cases the 90-100% of animals had the tumour, with the average latent period of tumour appearance after inoculation being the same both for irradiated and non-irradiated PIC-20 cells. After irradiation of parental DX-TK- cells with the highest dose of 15 Gy, the amount animals with tumour decreased by 70% and the average latent period of tumour appearance increased fivefold as compared with that for non-irradiated DX-TK- cells. The data obtained indicate that PIC-20 is highly radioresistant cells, which are able to proliferate both in semi-solid medium and in an animal organism even after radiation exposure to high doses.     

Mutability of the liver cells in the progeny of irradiated male rats

The descendants of the first generation of radiation exposed rat males exhibited an increased sensitivity of liver cells to the mutagenic effect of ionizing radiation as was determined by the incidence of chromosome aberrations. It is assumed that the stability of the hereditary apparatus of these animal cells is diminished due to the presence therein of radiation induced injuries of mutation and epigenomic origin at a heterozygous condition.     

Long-term effects of irradiation in the progeny of mammalian cells

It is shown that gamma-irradiation has remote consequences for mammalian cells cultivated in vitro. Many generations in the progeny of cells surviving acute and chronic irradiation at high and low doses are characterized by a number of abnormalities, including delayed cell death, the formation of micronuclei and giant cells, an increased frequency of sister chromatid exchanges, a reduced potential for repair, the loss of adaptive response, and increased radiosensitivity. These phenomena are regarded as manifestations of genomic instability induced by ionizing radiation.     

Relation of postradiation superovulation to the development of embryos and progeny of Wistar rats

In experiments with 470 primigravida Wistar rats and 2248 kid rats of the first generation a study was made of the relationship between the postirradiation superovulation and viability of embryos and offspring of females subjected to whole-body gamma-irradiation with doses of 1 to 4 Gy. The extent and durability of superovulation are a function of radiation dose while the superovulation itself is accompanied by an increased pre- or post-implantation death of embryos and a decreased postnatal survival of the offspring.     

DNA strand scission and its repair following exposure of cells to inhibitors of oxidative phosphorylation

Mouse L1210 leukemia and HeLa cells exposed to 2,4-dinitrophenol, oligomycin and rotenone under conditions which led to depletion of ATP pools exhibit DNA damage expressed as irreversible DNA strand separation in alkali. Removal of the agents allows both the repletion of ATP pools and repair of DNA damage.     

Clonogenicity of the progeny of surviving cells after irradiation

The clonogenic potential of the progeny of irradiated cells was tested in vitro by replating irradiated cultures after various times, allowing between five and over 25 subsequent divisions to take place after irradiation. Whereas the plating efficiency of surviving Chinese hamster cells was not decreased, in C3H10T1/2 cells a dose-dependent but slight decrease in plating efficiency was observed even after the longest follow-up period. These data do not contradict the prevalent hypothesis in radiobiology that the proliferation potential of a clonogenic cell surviving after irradiation is not significantly different from that of a non-irradiated cell.     

Assembly of Rickettsia tsutsugamushi progeny in irradiated L cells.

In the assembly of Rickettsia tsutsugamushi progeny in irradiated L cells, nascent forms first appear as undemarcated foci in the host cell granular cytoplasm, in which electron-lucent filamentous (f) and electron-dense granular (g) areas differentiate. Morphological observations indicated that the assembly involves formation of a filamentous network in the f area, manufacture of rickettsial ribosomes in the g area, and formation of mildly electron-dense fuzzy zones, along which a double membrane assembles.     

Delayed reproductive death and ROS levels in the progeny of irradiated melanoma cells

The cell death and survival of proliferating (clonogenic) cells were investigated in two human melanoma cell lines to assess the optimal conditions for preparation of apoptotic bodies from melanoma cells. After 50 J/m2 UVB+UVC the maximal levels of apoptotic cells assayed by Trypan blue staining, nucleosomal DNA fragmentation, MTT, and TUNEL tests were observed within 2-3 d of radiation. In 100 Gy gamma-irradiated cultures these apoptosis indicators were delayed for up to 3 weeks. In addition, clonogenic cells were observed only in exponentially growing cultures irradiated with UV at high cell density but not in gamma-irradiated cultures. The response of melanoma cultures after high UV radiation doses contrasted to the response in lethally gamma-irradiated cultures. UV-irradiated melanoma cultures were recovered within two weeks. Most of the clonogenic cells in the recovered colonies contained micronuclei. ROS levels determined by DCF fluorescence and a modified MTT test were also normalized obviously due to the extensive antioxidant defense system of melanoma cells. UV radiation of tumor cells might be the preferential method for preparation of apoptotic bodies. The presence of clonogenic cells in the suspension of apoptotic bodies from melanoma cells used for pulsing of dendritic cells with tumor antigens might compromise this protocol for preparation of cell vaccines.     

Persistent expression of morphological abnormalities in the distant progeny of irradiated cells

The phenomenon of delayed heritable lethal damage (often referred to as ``lethal mutations') in the progeny of cells which survive irradiation is now well established, but little is known of the mechanism by which this cell death occurs. Current theories suggest a generalised genomic instability affecting all cells which leads to the production of some mutations which are lethal, or alternatively that a lethal mutation gene is activated, mutated or induced by radiation and leads to persistent and random cell death at high levels in the progeny. The aim of this study was to look at the morphology of progeny of irradiated cells at various times after irradiation to establish how widespread morphological abnormalities were in the population and whether there was any evidence that such abnormalities were clonal. Using two different cell lines, the results showed that morphological evidence possibly suggestive of apoptosis occurred in the cultures after all doses of radiation and up to 45 cell doublings after exposure. There was no evidence of a decrease in the numbers of damaged or dead cells in colonies with number of divisions after irradiation, or with decreasing original radiation dose. There was a significant dose-dependent increase in the number of cells with microvilli for both cell lines. The dose-dependency of this effect did not change with number of divisions after irradiation. It is clear that morphological evidence of cellular damage persists for several generations after the initial exposure. The effects are widespread in the cell population, and their constancy over time argues strongly for a general instability and against a clonal mechanism, since clonal descendants should die out and leave undamaged survivors. The lack of evidence for necrosis or senescence together with many morphological changes in the cultures suggestive of apoptosis could indicate an active mechanism of cell death. It is concluded that survivor populations of irradiated cells from two widely different mammalian cell lines demonstrate an altered phenotype including gross morphological changes. These result in a higher probability that cell division will fail to yield two healthy progeny. Received: 22 January 1996 / Accepted in revised form: 24 September 1996     

Mutability of the germ cells of the progeny of irradiated male Drosophila

The increased frequency of random and radiation-induced mutation was registered in germ cells of drosophila irradiated male descendants of the first generation. The effect observed depended on of radiation dose delivered to parent males, test dose to progeny, type of mutation registered, and sex of the descendants under study.     

Chromosomal aberrations and DNA repair ability of in vitro irradiated white blood cells of monkeys previously exposed to total body irradiation

Six monkeys (Macaca fascicularis) were total-body-irradiated with 60Co (fractionated irradiation of 8 or 10 Gy). Blood samples were collected at different times post total-body irradiation, then in vitro irradiated in order to test whether a prior in vivo irradiation could affect the radiosensitivity of their leukocytes. We suggested in a preliminary report that the enhanced chromosomal radiosensitivity of in vivo irradiated monkeys could be correlated with a DNA repair deficiency (Guedeney et al., 1986). Chromosomal aberrations, the rate of initial strand breaks and their rejoining estimated using a fluorescent assay for DNA unwinding were chosen as the endpoints in this more extensive study. We observed that the yield of dicentrics induced by a subsequent in vitro irradiation was lower than that scored in unirradiated monkeys in few cases (6/22) whereas the number of acentrics was found to be modified in 16 of the 22 samples. An altered DNA repair ability was observed in most but not all blood samples tested. Thus, in view of such intra-individual variability, the results of this more extensive study lead us to conclude that a previous total-body irradiation does not alter the gamma-induced chromosome aberrations and DNA repair ability in a reproducible manner.     

Transmission of genomic instability from a single irradiated human chromosome to the progeny of unirradiated cells

Ionizing radiation can induce chromosome instability that is transmitted over many generations after irradiation in the progeny of surviving cells, but it remains unclear why this instability can be transmitted to the progeny. To acquire knowledge about the transmissible nature of genomic instability, we transferred an irradiated human chromosome into unirradiated mouse recipient cells by microcell fusion and examined the stability of the transferred human chromosome in the microcell hybrids. The transferred chromosome was stable in all six microcell hybrids in which an unirradiated human chromosome had been introduced. In contrast, the transferred chromosome was unstable in four out of five microcell hybrids in which an irradiated human chromosome had been introduced. The aberrations included changes in the irradiated chromosome itself and rearrangements with recipient mouse chromosomes. Thus the present study demonstrates that genomic instability can be transmitted to the progeny of unirradiated cells by a chromosome exposed to ionizing radiation, implying that the instability is caused by the irradiated chromosome itself and also that the instability is induced by the nontargeted effect of radiation.     

Assessment of DNA damage and repair in Mycobacterium terrae after exposure to UV irradiation

AIM: Ultraviolet (UV) irradiation for drinking water treatment was examined for inactivation and subsequent dark and photo-repair of Mycobacterium terrae. METHODS AND RESULTS: UV sources tested were low pressure (monochromatic, 254 nm) and medium pressure (polychromatic UV output) Hg lamps. UV exposure resulted in inactivation, and was followed by dark or photo-repair experiments. Inactivation and repair were quantified utilizing a molecular-based endonuclease sensitive site (ESS) assay and conventional colony forming unit (CFU) viability assay. Mycobacterium terrae was more resistant to UV disinfection compared to many other bacteria, with approximately 2-log reduction at a UV fluence of 10 mJ cm(-2) ; similar to UV inactivation of M. tuberculosis. There was no difference in inactivation between monochromatic or polychromatic UV lamps. Mycobacterium terrae did not undergo detectable dark repair. Photo-repair resulted in recovery from inactivation by approximately 0.5-log in less than 30 min for both UV lamp systems. CONCLUSIONS: Mycobacterium terrae is able to photo-repair DNA damage within a short timeframe. The number of pyrimidine dimers induced by UV light were similar for Escherichia coli and M. terrae, however, this similarity did not hold true for viability results. SIGNIFICANCE AND IMPACT OF THE STUDY: There is no practical difference between UV sources for disinfection or prevention of DNA repair for M. terrae. The capability of M. terrae to photo-repair UV damage fairly quickly is important for wastewater treatment applications where disinfected effluent is exposed to sunlight. Finally, molecular based assay results should be evaluated with respect to differences in the nucleic acid content of the test micro-organism.     

The effects of benzamide ADP-ribosyl transferase inhibitors on cell survival and DNA strand-break repair in irradiated mammalian cells

We have recently shown that 3-acetamidobenzamide (3-AAB), a highly effective inhibitor of ADP-ribosyl transferase (ADPRT), can act as a post-irradiation (electrons) sensitizer on the mouse lymphoma cell lines L5178Y R and S. We have now shown that this compound sensitizes human derived skin fibroblasts but to a lesser extent. Fibroblasts derived from normal, Friedreich's ataxia, and ataxia-telangiectasia individuals were equally sensitized by 3-AAB to electron radiation. 3-AAB was also effective in sensitizing the mouse lymphoma lines to fast neutron irradiation. In addition DNA strand break repair was retarded as had been found after electron irradiation. 3-Nitrobenzamide is structurally a potentially dual action radiation sensitizer with electron affinic and ADPRT inhibitory properties. It is a weaker inhibitor of ADPRT compared to 3-AAB, and results in a smaller sensitization of mouse lymphoma cells in air. However, a much greater sensitization is achieved in anoxia. This greater sensitization appears to be a synergistic rather than an additive combination of its electron affinic and ADPRT inhibitory properties.