Human lymphocytes exposed to low doses of X-rays are less susceptible to radiation-induced mutagenesis

Human lymphocytes exposed to low doses of X-rays become refractory to the subsequent induction of chromosomal damage by high doses of radiation (Shadley and Wolff, 1987). The current study was designed to test the effect of pretreatment of human T-lymphocytes with a low dose of X-rays on the induction of mutations at the hprt locus by a subsequent challenge dose. When cells were exposed to 1 cGy X-rays 24 h after phytohemagglutinin stimulation, the yield of mutations induced by a 300 cGy X-ray dose given 16 h later was reduced by approximately 70% from the control level of X-ray-induced mutations. This indicates that this previously described adaptive response to low dose X-rays also results in lymphocytes becoming refractory to the induction of gene mutations.     

Caffeine alone causes DNA damage in Chinese hamster ovary cells

Caffeine has been shown to enhance the lethal effect of DNA-damaging agents in mammalian cells, and the potentiation by caffeine of this effect is generally interpreted as the result of inhibition by caffeine of the repair of damaged DNA. However, the mechanism by which caffeine enhances the lethal effect of DNA-damaging agents has not yet been elucidated. During studies on the effect of caffeine on DNA repair, we found by alkaline elution analysis that caffeine alone produced DNA strand breaks or alkali labile sites in Chinese hamster ovary cells. The amount of DNA breakage or alkali labile sites depended on the concentration of caffeine. We propose that DNA breakage induced by caffeine may be involved in the enhancement of the lethal effect of DNA-damaging agents.     

DNA damage in cells exposed to ionizing radiation

Ionizing radiation induces variety of structural lesions in DNA of irradiated organisms. Their formation depends largely on the degree of cell oxygenation, the level of endogenous antioxidants, on DNA-protein complexes and compactization of DNA in the chromatin and activity of DNA repair systems. All ionizing radiation-induced DNA lesions can arbitrarily be divided into two groups. Group 1 includes singly damaged sites (single-sites): base modification, single-strand breaks, alkaline-labile sites (including a basic sites). Group 2 contains: locally multiply damaged sites (clustered lesions), double-strand breaks, intermolecular cross-links. The yields of lesions of group 2 increases with high linear energy transfer of radiation and these lesions play a dominant role in the radiation death, formation of chromosome and gene mutations, cell transformation.     

Adaptive response to chromosome damage in cultured human lymphocytes primed with low doses of X-rays

Human lymphocytes exposed to 0.02 Gy of X-rays in the G1 but not the G0 phase became less susceptible to the induction of chromosome aberrations of the chromosome type by subsequent exposure to 3 Gy of X-rays. The induction of chromatid-type aberrations was not affected by the pretreatment with the priming dose. The expression of this adaptive-type response was transitory, being maximum at 5 h, and disappeared at 9 h after the initial low-dose exposure. Cell-cycle analysis excluded the possibility of a spurious consequence of differential cell-cycle progression.     

DNA damage responses at low radiation doses

Increased cell killing after exposure to low acute doses of X rays (0-0.5 Gy) has been demonstrated in cells of a number of human tumor cell lines. The mechanisms underlying this effect have been assumed to be related to a threshold dose above which DNA repair efficiency or fidelity increases. We have used cells of two radioresistant human tumor cell lines, one that shows increased sensitivity to low radiation doses (T98G) and one that does not (U373), to investigate the DNA damage response at low doses in detail and to establish whether there is a discontinuous dose response or threshold in activation of any important mediators of this response. In the two cell lines studied, we found a sensitive, linear dose response in early signaling and transduction pathways between doses of 0.1 and 2 Gy with no evidence of a threshold dose. We demonstrate that ATM-dependent signaling events to downstream targets including TP53, CHK1 and CHK2 occur after doses as low as 0.2 Gy and that these events promote an effective damage response. Using chemical inhibition of specific DNA repair enzymes, we show that inhibition of DNA-PK-dependent end joining has relatively little effect at low (<1 Gy) doses in hyper-radiosensitive cells and that at these doses the influence of RAD51-mediated repair events may increase, based on high levels of RAD51/BRCA2 repair foci. These data do not support a threshold model for activation of DNA repair in hyper-radiosensitive cells but do suggest that the balance of repair enzyme activity may change at low doses.     

Radiosensitization of Chinese hamster cells by oxygen and misonidazole at low X-ray doses

The radiosensitization of Chinese hamster V79 cells in vitro by air and misonidazole at low X-ray doses (0.2-6.0 Gy) had been studied. These survival data, together with high-dose data, were fitted to the linear quadratic model ln S = -(alpha D + beta D2), deriving estimates of alpha and beta by six different methods to illustrate the influence of the statistical treatment on the values so derived. This in vitro study clearly demonstrated that the survival parameters alpha and beta are dependent to some degree on the method of analysis of the raw survival data; however, their ratios, the values of oxygen enhancement ratios (OERs) and radiosensitizer enhancement ratios (SERs) derived from the different methods, are similar. All methods of analysis give reduced OERs at low radiation doses for combined low- and high-dose X-ray data. However, the OERs are still appreciably high, ranging from 2.45 to 2.50 for an oxic dose of 2 Gy. All methods of analysis gave reduced SERs at low doses for combined low and high X-ray dose data for hypoxic cells irradiated in 1 mmol dm-3 misonidazole. At survival levels corresponding to doses of 2 Gy in the presence of 1 mmol dm-3 misonidazole and SERs ranged from 1.2 to 1.5.     

Enhanced cell killing, inhibition of recovery from potentially lethal damage and increased mutation frequency by 3-aminobenzamide in Chinese hamster V79 cells exposed to X-rays

X-ray induced potentially lethal damage and its inhibition by the aromatic amide 3-aminobenzamide have been investigated in Chinese hamster V79 cells. 3-Aminobenzamide (3-AB) is a known inhibitor of polyadenosine diphosphoribose synthetase. With increasing concentrations of 3-AB an increasing inhibition of PLD repair was observed. Little inhibition of PLD repair was seen when 3-AB was added 3 h following irradiation. Utilizing the 6-thioguanine mutation assay, the effect of poly(ADP-R) synthetase inhibition under conditions of PLD repair upon mutation frequency were also studied. A large increase in mutation frequency following 24 h post-irradiation recovery in the presence of 3-AB was seen. These results favour a possible role of 3-AB in preventing repair by facilitating early damage fixation before repair can occur, simultaneously reducing G2-arrest.     

DNA damage and repair in embryonic mouse limbs exposed to teratogenic doses of methylnitrosourea

The alkaline elution assay was used to monitor DNA single-strand breaks in embryonic tissue following exposure to the DNA-damaging teratogen N-methyl-N-nitrosourea (MNU, CAS No. 694-93-5). An animal model was developed in which nearly every fetus exposed to the highest dose of MNU had malformations of the hindlimbs while the fetuses exposed to the lowest dose of MNU had none. Hindlimbs pooled within litters were analyzed for DNA single-strand breaks by alkaline elution conducted at rapid (0.35 ml/min) and slow (0.35 ml/min) speeds. Breaks in the DNA of hindlimbs exposed to teratogenic doses of MNU were readily detected by alkaline elution only if slower speeds were used in the assay. Using the more sensitive procedure, DNA breakage was monitored over a 24-h period. DNA breakage peaked in the MNU-exposed hindlimbs in a dose-dependent manner 4 h after injection. While the elution profiles of hindlimbs exposed to the lower doses of MNU returned to control levels 8 h after injection, single-strand breaks persisted in the hindlimbs exposed to the highest dose of MNU for at least 20 h. These latter data suggest that the highly teratogenic dose of MNU induced DNA damage that was more slowly repaired than that produced at lower doses, possibly by saturation of DNA repair systems. Although some necrosis did occur in hindlimbs exposed at teratogenic dose levels, it was not severe and it did not appear to influence the alkaline elution results. These experiments show that alkaline elution is a sensitive assay for the detection of DNA damage in embryonic tissues.     

DNA damage and DNA repair in cultured human cells exposed to chromate

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.     

DNA damage and repair in Chinese hamster fibroblasts exposed to gamma and secondary radiation generated by 70 GeV protons

The cytochemical study of DNA damage and repair in a Chinese hamster fibroblast culture exposed to gamma-rays and secondary radiation from 70 GeV protons showed no significant differences between the two types of radiation.     

The characteristics of the realization of cytogenetic damage in mammalian and plant cells exposed to low doses of radiation]

A complicated character of the cytogenetic injury dependence upon radiation dose was revealed after low-level gamma irradiation of Vicia faba seedlings and Chinese hamster fibroblasts. The dependence was linear with low-level secondary exposure to 70 GeV protons. The authors discuss a threshold nature of induction of the cytogenetic damage repair responsible for a high outcome of damages under the effect of low-level gamma radiation.     

Oxidative damage of Chinese hamster fibroblasts induced by t-butyl hydroperoxide and by X-rays

The aim of this study was to investigate the mechanism(s) of X-ray-mediated cell damage in comparison to mechanism(s) of organic hydroperoxide cytotoxicity and to find the main targets for the two different kinds of cell inactivation. Damage of Chinese hamster fibroblasts induced by tert-butyl hydroperoxide (t-BHP) or X-irradiation was measured by the colony-formation assay and the average single colony volume. DNA double-strand breaks (dsb) were determined by constant-field gel electrophoresis. The contents of peroxides, of SH-groups and the size of inactivated cells were tested for oxidative modifications.Oxidative damage of fibroblasts induced by t-BHP or by X-rays inhibits cell proliferation. Simultaneously, irradiation causes an increase of DNA dsb with the dose, while incubation with t-BHP yields only a very few DNA dsb. Neither chemically induced oxidation nor irradiation significantly changed the amount of membrane lipid peroxides. Oxidation with t-BHP but not irradiation leads to a loss of the membrane SH-groups and to an increase of cell diameter.The similar decrease of cell proliferation can be caused by DNA dsb without detectable membrane damage (X-radiation) as by membrane damage with nearly no DNA dsb (chemically induced oxidative stress).     

Telomeric associations in cigarette smokers exposed to low levels of X-rays

Telomeric association (TA), i.e. fusion of chromosomes by their telomeres, predisposes a cell to genetic instability. Because of this we investigated the effect of X-rays exposure and cigarette smoking on the frequency of TA in peripheral blood lymphocytes of exposed individuals, in order to determine if TA can be a chromosomal marker in populations exposed to these carcinogens and if there is an synergistic effect between both agents. We found that the exposed groups show a greater percentage of TA when compared with the control group (P<0.001). However, although the percentage of metaphases with TA in the group with combined exposure (12.6%) was greater than in the others exposed groups (P<0.05), this value was less than the sum of the two individual effects (15.1%). Our results suggest that probably there is not an additive or synergistic effect between X-rays and smoking, and that TA may be a useful cytogenetic marker for evaluating populations exposed to mutagens.     

DNA damage and repair in mammalian cells exposed to p-hydroxyphenylpyruvic acid

Tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine metabolism in which the fumarylacetoacetate hydrolase enzyme is defective. This disease is clinically heterogeneous and a chronic and acute form is discerned. Characteristic of the chronic form is the development of cellular hepatocarcinoma. Although p-hydroxyphenylpyruvic acid (pHPPA) is used as one of the diagnostic markers of this disease, it was suggested that it is unlikely to be involved in the pathophysiology of HT1 as it is present in other disorders that does not have hepatorenal symptoms. It was the aim of this study to investigate the possible effect of pHPPA on DNA damage and repair in mammalian cells. The comet assay was used to establish the genotoxicity of pHPPA in human peripheral blood lymphocytes and isolated rat hepatocytes after their exposure to pHPPA. At first glance the damage to DNA caused by pHPPA seemed reparable in both cell types, however, after challenging the DNA repair capacity of metabolite-treated cells with treatment with H(2)O(2), a marked impairment in the DNA repair capability of these cells was observed. We suggest that the main effect of pHPPA is the long-term impairment of the DNA repair machinery rather than the direct damage to DNA and that this effect of pHPPA, together with the other characteristic metabolites, e.g., FAA and MAA, causes cellular hepatocarcinoma to develop in the chronic form of HT1.     

Processing of abasic DNA clusters in hApeI-silenced primary fibroblasts exposed to low doses of X-irradiation

Clustered damage in DNA includes two or more closely spaced oxidized bases, strand breaks or abasic sites that are induced by high- or low-linear-energy-transfer (LET) radiation, and these have been found to be repair-resistant and potentially mutagenic. In the present study we found that abasic clustered damages are also induced in primary human fibroblast cells by low-LET X-rays even at very low doses. In response to the induction of the abasic sites, primary fibroblasts irradiated by low doses of X-rays in the range 10–100 cGy showed dose-dependent up-regulation of the DNA repair enzyme, ApeI. We found that the abasic clusters in primary fibroblasts were more lethal to cells when hApeI enzyme expression was down-regulated by transfecting primary fibroblasts with hApeI siRNA as determined by clonogenic survival assay. Endonuclease activity of hApeI was found to be directly proportional to hApeI gene-silencing efficiency. The DNA repair profile showed that processing of abasic clusters was delayed in hApeI-siRNA-silenced fibroblasts, which challenges the survival of the cells even at very low doses of X-rays. Thus, the present study is the first to attempt to understand the induction of cluster DNA damage at very low doses of low-LET radiation in primary human fibroblasts and their processing by DNA repair enzyme ApeI and their relation with the survival of the cells.     

Metal-induced DNA damage and repair in human diploid fibroblasts and Chinese hamster ovary cells

Cloning efficiency and DNA strand breaks induction were compared in human diploid fibroblasts (HSBP) and chinese hamster ovary (CHO) cells treated with various metal salts. Cadmium (Cd2+), nickel (Ni2+) and chromate (Cr2O7) reduced the cloning efficiency of HSBP cells more than that of CHO cells whereas the reverse was true after treatment with mercury (Hg2+), manganese (Mn2+) and cobalt (Co2+). The effects on cloning efficiency did not consistently correlate with DNA strand breaking activity as all metals except Cr(VI) were more effective at producing DNA strand breaks in CHO cells than in human cells. The differential responses of the two cell types was shown to be only partially due to differences in cellular uptake of metals. DNA breaks induced in human cells by Hg2+ and Cr2O7 were shown most likely to be alkaline labile sites rather than true strand breaks since no damage was detected in a nick translation assay which measures the amount of free 3'-OH terminals. Damage induced by Mn2+ and Co2+, however, appeared to be comprised at least in part by true DNA strand breaks. DNA damage was also induced in HSBP cells following treatment with selenium but only in the presence of reduced glutathione. These studies indicate that DNA damage is not as major a consequence following some metal treatments in human cells as it appears to be in rodent cells. This suggests that rodent models for risk estimation of metal-induced tumorigenesis may not always be appropriate for extrapolation to humans.