Photoreactivation of damage induced by ionizing radiation in yeast cells

Summary Experimental data on photoreactivation of damage induced by ionizing radiation in yeast cells are presented. The value of photoreactivation was found to be the highest for the following conditions predicted by us as optimum ones: large volume of irradiated suspension, hypoxia and high energy sparsely ionizing radiation. A comparison of data for yeast and bacterial cells shows that Cerenkov emission from ionizing radiation may produce photoreactivated pyrimidine dimers in both prokaryotic and eukaryotic cell systems.     

Modification of high LET radiation-induced damage and its repair in yeast by hypoxia.

The lethal response of a diploid yeast strain BZ34 to densely ionizing radiations from the reaction 10B(n, alpha)7 Li was studied. The values for relative biological effectiveness (r.b.e.) and oxygen enhancement ratio (o.e.r.) for this radiation compare favourably with the data obtained with charged particles on the same strain of yeast. Recovery from potentially lethal damage was also studied by post-irradiation holding under non-nutrient conditions. In order to understand the role of oxygen in the recovery process, the investigation covered the following treatment regimens: (a) aerobic irradiation and aerobic holding (A-A), (b) aerobic irradiation and hypoxic holding (A-H), (c) hypoxic irradiation and hypoxic holding (H-H) and (d) hypoxic irradiation and aerobic holding (H-A). It has been found that the presence of oxygen is essential for recovery from the damage induced by both gamma rays and high linear energy transfer (LET) radiations. The extent of recovery was larger for gamma-induced damage than for damage induced by high LET radiation (alpha + 7Li) for the A-A condition. In the H-H condition, while only a slight recovery was seen for gamma-induced damage, it was totally absent for high LET damage. For the modality A-H, it was found that there is not recovery from the sparsely ionising gamma radiation-induced damage. The implications of these results for the treatment of malignant tumours by radiotherapy are briefly discussed.     

Induction and repair of DNA damage as measured by the Comet assay and the yield of somatic mutations in gamma-irradiated tobacco seedlings

The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values ( +/- S.E.) significantly increased from 1.08 +/- 0.10 (control) to 20.26 +/- 1.61 microm (10Gy). Nuclei isolated from leaves 24h after irradiation expressed tail moment values that were not significantly different from the control (2.08 +/- 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented.     

Recovery from lethal and mutagenic damage during postirradiation holding and low-dose-rate irradiations of cultured hamster cells

Survival and mutation to thioguanine resistance were measured in V79-4 hamster cells grown to plateau phase without refeeding and irradiated with 60Co gamma rays. The effects of low-dose-rate irradiation and of postirradiation holding on recovery from gamma-ray damage leading to these two responses were also studied. The responses of these plateau (extended G1)-phase cells to acute irradiation were similar to those we previously found for exponentially growing cells, including the linear relationship between induced mutant frequency and (log) surviving fraction. Irradiation at low dose rate (0.34 rad/min) considerably reduced both the lethal and mutagenic effects of given doses of gamma rays, but the linear mutation-survival relationship was approximately the same as for acute irradiation. In contrast, cells given a 5-hr holding period after acute irradiation showed the anticipated recovery from potentially lethal damage but no recovery from damage leading to mutation. These results are discussed in terms of previously proposed cellular repair processes (sublethal damage repair and potentially lethal damage repair) and the possibility that the radiation damage leading to lethality is different from mutagenic damage.     

Cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with accelerated 56Fe ions

We examined cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with high-energy 56Fe ions. Cells were irradiated with graded doses of 56Fe ions (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The survival curves for cells plated 1 h after irradiation (immediate plating) showed little or no shoulder. However, the survival curves for cells plated 24 h after irradiation (delayed plating) had a small initial shoulder. The RBE for 56Fe ions compared to 137Cs gamma rays was 1.99 for immediate plating and 2.73 for delayed plating at the D10. The repair ratio (delayed plating/immediate plating) was 1.67 for 137Cs gamma rays and 1.22 for 56Fe ions. The dose-response curves for initially measured and residual chromatid fragments detected by the Calyculin A-mediated premature chromosome condensation technique showed a linear response. The results indicated that the induction frequency for initially measured fragments was the same for 137Cs gamma rays and 56Fe ions. On the other hand, approximately 85% of the fragments induced by 137Cs gamma rays had rejoined after 24 h of postirradiation incubation; the corresponding amount for 56Fe ions was 37%. Furthermore, the frequency of chromatid exchanges induced by gamma rays measured 24 h after irradiation was higher than that induced by 56Fe ions. No difference in the amount of chromatid damage induced by the two types of radiations was detected when assayed 1 h after irradiation. The results suggest that high-energy 56Fe ions induce a higher frequency of complex, unrepairable damage at both the cellular and chromosomal levels than 137Cs gamma rays in the target cells for radiation-induced lung cancers.     

A fluorescence approach of the gamma radiation effects on gramicidin A inserted in liposomes.

The fluorescence of tryptophan residues of gramicidin A (gA), bound to phosphatidylcholine liposomes contains valuable information about local changes in the environment of the molecule induced by gamma radiation. With this work, we aim to demonstrate that the gamma radiation effect on the peptide involves the action of free radicals, derived from water radiolysis and the process of lipid peroxidation. Basically, the methodology consists of the analysis of UV and fluorescence emission spectra of the peptide liposome complexes under control conditions and upon gamma irradiation. Free radical production was impaired by the removal of molecular oxygen or the presence of ethanol in the liposome suspension. The intensity of the tryptophan fluorescence was recorded as a function of the gamma radiation dose in the range of 0-250 Gy and the data were fitted with a single decay exponential function containing an additional constant term (named residual fluorescence). The correlation between the decrease in tryptophan fluorescence emission (D(c) = 80 +/- 10 Gy) and increase in gamma radiation dose indicates the partial damage of the tryptophan side chains of gA. O(2) removal or ethanol addition partially reduced the decay of the tryptophan fluorescence emission involving an indirect action of gamma radiation via a water radiolysis mechanism. The residual fluorescence emission (A(0)) increases in O(2)-free buffer (98 +/- 13) and in 10% ethanol-containing buffer (74 +/- 34) compared to control conditions (23 +/- 5). Varying the dose rate between 1-10 Gy/min at a constant dose of 50 Gy, an inverse dose-rate effect was observed. Thus, our study provides evidence for the lipid peroxidation effect on the tryptophan fluorescence. In conclusion, this article sustains the hypothesis of the connection between the lipid peroxidation and structural changes of membrane proteins induced by gamma radiation. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

UV-type damage associated with ionizing radiation: a review

The induction of UV-type damage by ionizing radiation in repair deficient strains of E. coli is reviewed. Both photoreactivable and non-photoreactivable types of damage can be observed. The induction of UV-type damage is largely independent of the presence of free-radical reactive agents (e.g. oxygen and thiols), but is dependent upon the energy of the photon--or electron--beam used, the radiation geometry and the optical absorbance of the extracellular medium. On the basis of calculations and experimental evidence, it is clear that one mechanism whereby such damage arises is through the generation of Cerenkov emission. However, small yields of UV-type damage can be produced using X-rays whose energy is below the threshold for production of Cerenkov emission. In this instance, the damage induction mechanism is thought to involve a direct excitation process.     

Liquid holding recovery in stationary and log phase cultures of diploid yeast exposed to gamma and alpha radiations

Summary The kinetics of liquid-holding recovery (LHR) in diploid yeast after gamma and alpha irradiation is studied. In case of stationary phase culture the rate and extent of LHR is found to be greater for gamma-ray-induced damage than for alpha-ray-induced damage. At 10% survival level, the half-time for recovery is 5.2 h for gamma-ray damage and 12 h for alpha-ray damage. Further, while the recovery factor for alpha damage reaches saturation at 5% survival level, that for gamma damage continues to increase as survival level decreases. Oxygen is required for the recovery process during LH after gamma irradiation. The cells can recover to the same extent from both oxygen-dependent and oxygen-independent components of damage. Log phase cells containing a high per cent of budding cells, however, exhibit negative liquid holding effect after gamma irradiation.     

Physiological responses of the halophilic archaeon Halobacterium sp. strain NRC1 to desiccation and gamma irradiation

We report that the halophilic archaeon Halobacterium sp. strain NRC-1 is highly resistant to desiccation, high vacuum and ⁶⁰Co gamma irradiation. Halobacterium sp. was able to repair extensive double strand DNA breaks (DSBs) in its genomic DNA, produced both by desiccation and by gamma irradiation, within hours of damage induction. We propose that resistance to high vacuum and ⁶⁰Co gamma irradiation is a consequence of its adaptation to desiccating conditions. Gamma resistance in Halobacterium sp. was dependent on growth stage with cultures in earlier stages exhibiting higher resistance. Membrane pigments, specifically bacterioruberin, offered protection against cellular damages induced by high doses (5 kGy) of gamma irradiation. High-salt conditions were found to create a protective environment against gamma irradiation in vivo by comparing the amount of DSBs induced by ionizing radiation in the chromosomal DNA of Halobacterium sp. to that of the more radiation-sensitive Escherichia coli that grows in lower-salt conditions. No inducible response was observed after exposing Halobacterium sp. to a nonlethal dose (0.5 kGy) of gamma ray and subsequently exposing the cells to either a high dose (5 kGy) of gamma ray or desiccating conditions. We find that the hypersaline environment in which Halobacterium sp. flourishes is a fundamental factor for its resistance to desiccation, damaging radiation and high vacuum.     

Human epithelial teratocarcinoma cells (P3): radiobiological characterization, DNA damage, and comparison with other rodent and human cell lines

Survival parameters and immediate DNA damage induced by 60Co gamma rays, 50-kVp X rays, and Janus fission-spectrum neutrons in human epithelial P3 cells (derived from an embryonic teratocarcinoma) are compared with those for Chinese hamster lung V79 cells. DNA damage caused by X and gamma irradiation, measured by alkaline elution methods, is the same in both cell types, whereas the P3 cells are about two times more sensitive (as measured by Do ratios of the final survival curve slope) to the lethal effects of these radiations than are the V79 cells. Human P3 cells are also more sensitive to the lethal effects of fission-spectrum neutrons than V79 cells. Survival experiments with split radiation doses and hypertonic salt treatment indicate that both P3 cells and V79 cells can recover from radiation-induced damage efficiently.     

Genome‐wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato

Genome‐wide mutations induced by ethyl methanesulfonate (EMS) and gamma irradiation in the tomato Micro‐Tom genome were identified by a whole‐genome shotgun sequencing analysis to estimate the spectrum and distribution of whole‐genome DNA mutations and the frequency of deleterious mutations. A total of ~370 Gb of paired‐end reads for four EMS‐induced mutants and three gamma‐ray‐irradiated lines as well as a wild‐type line were obtained by next‐generation sequencing technology. Using bioinformatics analyses, we identified 5920 induced single nucleotide variations and insertion/deletion (indel) mutations. The predominant mutations in the EMS mutants were C/G to T/A transitions, while in the gamma‐ray mutants, C/G to T/A transitions, A/T to T/A transversions, A/T to G/C transitions and deletion mutations were equally common. Biases in the base composition flanking mutations differed between the mutagenesis types. Regarding the effects of the mutations on gene function, >90% of the mutations were located in intergenic regions, and only 0.2% were deleterious. In addition, we detected 1 140 687 spontaneous single nucleotide polymorphisms and indel polymorphisms in wild‐type Micro‐Tom lines. We also found copy number variation, deletions and insertions of chromosomal segments in both the mutant and wild‐type lines. The results provide helpful information not only for mutation research, but also for mutant screening methodology with reverse‐genetic approaches.     

Neutron radiation can activate K-ras via a point mutation in codon 146 and induces a different spectrum of ras mutations than does gamma radiation.

Neutron radiation is known to produce tumors in animals and cause cell transformation. We have developed a protocol to efficiently induce thymic lymphomas in RF/J mice by a single acute dose of neutron irradiation. Activated ras genes were detected in 17% (4 of 24) of the tumors analyzed. One of the tumors contained a K-ras gene activated by a point mutation in codon 146. Activating ras mutations at position 146 have not been previously detected in any known human or animal tumors. The spectrum of ras mutations detected in neutron radiation-induced thymic lymphomas was different from that seen in thymic lymphomas induced by gamma radiation in the same strain of mice. These results may have important implications for the mechanisms by which different types of radiation damage DNA.     

Biochemical alterations in human cells irradiated with alpha particles delivered by macro- or microbeams

Irradiation of individual cell nuclei with charged-particle microbeams requires accurate identification and localization of cells using Hoechst staining and UV illumination before computer-monitored localization of each cell. Using Fourier-transform infrared microspectroscopy (FT-IRM), we investigated whether the experimental conditions used for cell recognition induce cellular changes prior to irradiation and compared biochemical changes and DNA damage after targeted and nontargeted irradiation with alpha particles delivered by macro- or microbeams, using gamma radiation as a reference. Molecular damage in single HaCaT cells was studied by means of FT-IRM and comet assay (Gault et al., Int. J. Radiat. Biol. 81, 767-779, 2005). Hoechst 33342-stained HaCaT cells were exposed to single doses of 2 Gy (239)Pu alpha particles from a broad-beam irradiator, five impacted alpha particles from a microbeam irradiator, or 6 Gy gamma rays from (137)Cs, each of which resulted in about 5% clonogenic survival. FT-IRM of control cells indicated that Hoechst binding to nuclear DNA induced subtle changes in DNA conformation, and its excitation under UV illumination induced a dramatic shift of the DNA conformation from A to B as well as major DNA damage as measured by the comet assay. Comparison of the FT-IRM spectra of cells exposed to gamma rays or alpha particles specifically targeted to the nucleus, alpha particles from a broad-beam irradiator revealed spectral changes corresponding to all changes in constitutive bases in nucleic acids, suggesting oxidative damage in these bases, as well as structural damage in the deoxyribose-phosphate backbone of DNA and the osidic structure of nucleic acids. Concomitantly, spectral changes specific to protein suggested structural modifications. Striking differences in IR spectra between targeted microbeam- and nontargeted macrobeam-irradiated cells indicated greater residual unrepaired or misrepaired damage after microbeam irradiation. This was confirmed by the comet assay data. These results show that FT-IRM, together with the comet assay, is useful for assessing direct radiation-induced damage to nucleic acids and proteins in single cells and for investigating the effects of radiation quality. Significantly, FT-IRM revealed that Hoechst 33342 binding to DNA and exposure to UV light induce a dramatic change in DNA conformation as well as DNA damage. These findings suggest that fluorochrome staining should be avoided in studies of ionizing radiation-induced bystander effects based on charged-particle microbeam irradiation. An alternative cell nucleus recognition system that avoids nuclear matrix damage and its possible contribution to propagation of biological effects from irradiated cells to neighboring nontargeted cells needs to be developed.     

Mutagen sensitivity of human lymphoblastoid cells with a BRCA1 mutation in comparison to ataxia telangiectasia heterozygote cells

Our previous results indicated a close relationship between the presence of a BRCA1 mutation in lymphocytes and hypersensitivity for the induction of micronuclei by gamma irradiation and hydrogen peroxide (H(2)O(2)). Comparative investigations with the comet assay (single-cell gel electrophoresis) suggested a normal rate of damage removal and pointed to a disturbed fidelity of DNA repair as a direct or indirect consequence of a BRCA1 mutation. We now wanted to see whether similar results could be obtained with lymphoblastoid cell lines (LCLs) and whether such permanent cells are suitable as a model for the investigation of mechanisms involved in mutagen sensitivity. Our results show that LCLs with a BRCA1 mutation are also hypersensitive to the chromosome-damaging effects of gamma irradiation or H(2)O(2), as revealed by the micronucleus test. Interestingly, LCLs heterozygous for an ataxia telangiectasia (AT) mutation have similar characteristics as BRCA1 cells with respect to the induction and repair of DNA damage induced by either gamma irradiation or H(2)O(2). However, caffeine enhanced the induction of micronuclei by gamma irradiation only in normal and heterozygous AT cells but not in BRCA1 cells, thus indicating a difference in the pathways leading to mutagen sensitivity in cells with a BRCA1 or an AT mutation. Our results suggest that caffeine could be useful in discriminating AT heterozygotes from carriers of a BRCA1 mutation, as well as BRCA1 mutation carriers from normal individuals.     

Measurements of alkali-labile DNA damage and protein-DNA crosslinks after 2450 MHz microwave and low-dose gamma irradiation in vitro

In vitro experiments were performed to determine whether 2450 MHz microwave radiation induces alkali-labile DNA damage and/or DNA-protein or DNA-DNA crosslinks in C3H 10T(1/2) cells. After a 2-h exposure to either 2450 MHz continuous-wave (CW) microwaves at an SAR of 1.9 W/kg or 1 mM cisplatinum (CDDP, a positive control for DNA crosslinks), C3H 10T(1/2) cells were irradiated with 4 Gy of gamma rays ((137)Cs). Immediately after gamma irradiation, the single-cell gel electrophoresis assay was performed to detect DNA damage. For each exposure condition, one set of samples was treated with proteinase K (1 mg/ml) to remove any possible DNA-protein crosslinks. To measure DNA-protein crosslinks independent of DNA-DNA crosslinks, we quantified the proteins that were recovered with DNA after microwave exposure, using CDDP and gamma irradiation, positive controls for DNA-protein crosslinks. Ionizing radiation (4 Gy) induced significant DNA damage. However, no DNA damage could be detected after exposure to 2450 MHz CW microwaves alone. The crosslinking agent CDDP significantly reduced both the comet length and the normalized comet moment in C3H 10T(1/2) cells irradiated with 4 Gy gamma rays. In contrast, 2450 MHz microwaves did not impede the DNA migration induced by gamma rays. When control cells were treated with proteinase K, both parameters increased in the absence of any DNA damage. However, no additional effect of proteinase K was seen in samples exposed to 2450 MHz microwaves or in samples treated with the combination of microwaves and radiation. On the other hand, proteinase K treatment was ineffective in restoring any migration of the DNA in cells pretreated with CDDP and irradiated with gamma rays. When DNA-protein crosslinks were specifically measured, we found no evidence for the induction of DNA-protein crosslinks or changes in amount of the protein associated with DNA by 2450 MHz CW microwave exposure. Thus 2-h exposures to 1.9 W/ kg of 2450 MHz CW microwaves did not induce measurable alkali-labile DNA damage or DNA-DNA or DNA-protein crosslinks.     

Migration patterns of dendritic cells in the rat: comparison of the effects of gamma and UV-B irradiation on the migration of dendritic cells and Lymphocytes

To further define the underlying mechanisms of immune suppression induced by UV-B irradiation, we have examined the kinetics of homing patterns of in vitro UV-B-irradiated and gamma-irradiated-thoracic duct lymphocytes (TDL) compared to dendritic cells (DC). Our findings show that 111In-oxine-labeled TDL specifically home to the spleen, liver, lymph nodes, and bone marrow with subsequent recirculation of a large number of cells from the spleen to lymph nodes. In contrast, DC preferentially migrate to the spleen and liver with a relatively insignificant distribution to lymph nodes and an absence of subsequent recirculation. Splenectomy prior to cell injection significantly diverts the spleen-seeking DC to the liver but not to the lymph nodes, while the homing of TDL to lymph nodes is significantly increased. In vitro exposure of 111In-oxine labeled TDL to gamma irradiation does not significantly impair immediate homing to lymphoid tissues but inhibits cell recirculation between 3 and 24 hr. In contrast, gamma irradiation does not affect the tissue distribution of labeled DC, suggesting that DC are more radioresistant to gamma irradiation than TDL. Unlike the findings in animals injected with gamma-irradiated cells, UV-B irradiation virtually abolished the homing of TDL to lymph nodes and significantly reduced the homing of the spleen-seeking DC to the splenic compartment while a large number of cells were sequestered in the liver. The results of in vitro cell binding assay show that TDL, unlike DC, have the capacity to bind to high endothelial venules (HEV) within lymph node frozen sections while gamma and UV-B irradiation significantly inhibit the binding of TDL to lymph node HEV. These findings suggest that: (i) DC, unlike TDL, are unable to recirculate from blood to lymph nodes through HEV; (ii) although gamma irradiation impairs TDL recirculation, it does not affect DC tissue distribution; and (iii) UV-B irradiation impairs both TDL and DC migration patterns. We conclude that the lack of capacity of irradiated TDL to home to lymph nodes is due to damage to cell surface homing receptors and that the failure of DC to home to the lymph node microenvironment is related to the absence of HEV homing receptors on their cell surface.     

Mutant frequencies and spectra depend on growth state and passage number in cells cultured from transgenic lacZ-plasmid reporter mice

Transgenic mice harboring the lacZ gene within a plasmid that can be recovered and amplified in Escherichia coli, to establish mutant frequencies and spectra, have provided crucial insights into the relationships between mutations, cancer and aging in vivo. Here, we use embryonic fibroblasts from transgenic lacZ-plasmid reporter mice to determine the relationship between cell proliferation in culture and mutations induced by ultraviolet (UV) light. A single dose of 2.5J/m2 of UVC to actively proliferating cells caused an approximately eight-fold increase in mutant frequency 24 h after irradiation. Identically treated quiescent cells showed a two-fold increase in mutant frequency. Thus, whereas proliferation facilitated the acquisition of mutations, it was not an absolute requirement. Characterization of the UV-induced mutations indicated that the lower mutant frequency in quiescent cells was due mainly to a reduction in point mutations; size-change mutations, indicative of translocations or deletions, were relatively unaffected by the growth state of the cells. To investigate long-term genomic stability after UVC-induced damage, we monitored the lacZ locus in irradiated cells passaged for many generations in culture. The results indicated the emergence of jackpot mutations of rapidly changing frequency, most likely reflecting the successive emergence and decline of dominant cell clones during long-term culture. These findings show that the lacZ-plasmid locus is a valid reporter for studying induced mutations in short-term cultures of both quiescent and proliferating fibroblasts. In long-term cultures, the locus is less suitable for studying induced mutations owing to the instability of the cell population.     

Novel role of p53 in maintaining mitochondrial genetic stability through interaction with DNA Pol gamma

Mitochondrial DNA (mtDNA) mutations and deletions are frequently observed in cancer, and contribute to altered energy metabolism, increased reactive oxygen species (ROS), and attenuated apoptotic response to anticancer agents. The mechanisms by which cells maintain mitochondrial genomic integrity and the reason why cancer cells exhibit more frequent mtDNA mutations remain unclear. Here, we report that the tumor suppressor molecule p53 has a novel role in maintaining mitochondrial genetic stability through its ability to translocate to mitochondria and interact with mtDNA polymerase gamma (pol gamma) in response to mtDNA damage induced by exogenous and endogenous insults including ROS. The p53 protein physically interacts with mtDNA and pol gamma, and enhances the DNA replication function of pol gamma. Loss of p53 results in a significant increase in mtDNA vulnerability to damage, leading to increased frequency of in vivo mtDNA mutations, which are reversed by stable transfection of wild-type p53. This study provides a mechanistic explanation for the accelerating genetic instability and increased ROS stress in cancer cells associated with loss of p53.     

Cell Cycle Kinetics and Radiation-Induced Chromosomal Aberrations Studied with C14 and H3 Labels

Chinese hamster cells in vitro were double labeled with C(14)TdR and H(3)TdR. At the time of irradiation with Co(60) gamma rays (600 rad), the cells in the G(2) phase were labeled only with C(14), whereas cells in the late and middle S phases were labeled with both C(14) and H(3). The cells in early S phase were labeled only with H(3) and the G(1) cells were unlabeled. Samples were fixed at various time intervals following irradiation and the metaphases were analyzed for chromosomal damage. The phase in which the cell was located at the time of irradiation was determined by counting grains in the first and second layers of autoradiographic film. In both control and irradiated cells some G(1) cells divided prior to some of the cells which were in the S phase denoting mixing of the populations. The G(2) phase sustained three times more chromosomal damage than the S phase. Little difference in chromosomal damage was found between the G(1) and S phases or among the different parts of the S phase. Cells in G(2) sustained a mitotic delay of 4 hr, while the other phases sustained a delay of 2 to 3 hr. Chromatid and chromosome (dicentrics) exchanges were induced in G(1) cells but only chromatid exchanges were induced in S and G(2) cells; this is consistent with the hypothesis that the chromosome consists of two subunits which separate either slightly before or immediately as the cell enters the S phase.     

Adaptive response to mutagenesis and its molecular basis in a human T-cell leukemia line primed with a low dose ofγ-rays

The effect was studied of a low dose of-ray preexposure on the frequency and molecular spectrum of radiation-induced mutations at the hprt locus in a human T-cell leukemia line. When the cells were preexposed to 0.01 Gy of-rays, the yield of mutations induced by a subsequent 2-Gy challenge dose was reduced by 60%, compared with the 2 Gy of irradiation alone. The data of Southern blot analysis showed that 47% of the mutants induced by 2 Gy in the cells without low-dose preexposure were of the deletion or rearranged mutations type. In contrast, in the low-dose radioadapted cells the proportion of this type of 2-Gy-induced mutations decreased to 28%. This is close to the control level (22%) of spontaneous mutations. Our results confirm that a low dose of-ray preexposure leads to a decreased susceptibility to gene deletions and rearrangements after high-dose irradiation.