The induction by H 2 O 2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H 2 O 2 in suspension at 0°C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H 2 O 2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H 2 O 2 concentration was observed at low H 2 O 2 concentrations that reached a maximum at approximately 1 mM; at higher H 2 O 2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H 2 O 2 : cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H 2 O 2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H 2 O 2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H 2 O 2 where cell killing is observed by an unidentified mechanism.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle. Results on the quantification of DNA DSBs and SSBs in cells substituted with BrdU are also presented and differences from data published previously are discussed; it is suggested that quantitative measurement of alterations in the induction by radiation of damage in BrdU-substituted DNA may be hampered by changes induced in the physicochemical properties of the molecule that affect the behavior of DNA molecules during the assays and complicate the interpretation of the results obtained.

The effect of the DNA polymerase inhibitor β-arabinofuranosyladenine (araA) on radiation-induced damage was studied at the cell survival and chromosome level in unfed plateau-phase cultures of Chinese hamster ovary cells. At the cell survival level postirradiation treatment with araA fixed a form of radiation-induced potentially lethal damage, termed α-PLD. In the absence of araA treatment, repair of PLD resulted in the formation of the survival curve shoulder in immediately plated cells and in the increase in survival observed after delayed plating. The repair kinetics observed after delayed plating of plateau-phase cells or after delayed administration of 500 μM araA were similar, suggesting that both protocols assay similar lesions. AraA-mediated fixation reached a plateau at concentrations higher than 500 μM, indicating complete fixation of α-PLD. At the cytogenetic level, postirradiation treatment with araA at concentrations higher than 500 μM caused a complete inhibition of chromosome repair, as scored by premature chromosome condensation. In the absence of araA, the linearity of the dose-effect relationship for chromosome fragmentation obtained immediately after irradiation was preserved even after long repair times. The repair kinetics of chromosome damage measured in cells held postirradiation in the plateau phase were the mirror image of the repair kinetics for α-PLD. The half-time was 1 h in both cases and repair reached a plateau after about 4-6 h. AraA-mediated repair inhibition of chromosome damage was reversible, and a decrease in residual chromosome damage was observed after post-treatment incubation in araA-free conditioned medium. This persistent chromosome damage increased with increasing araA concentration and, as with PLD fixation, reached a plateau at about 500 μM. These results suggest that repair and araA-mediated fixation of α-PLD have their counterparts at the chromosome level as indicated by the similar repair kinetics and inhibition/fixation characteristics obtained for α-PLD and chromosome damage. This relationship implies a correlation between repair at the DNA and the chromosome level and suggests that DNA polymerization is required for the repair of chromosome damage.

The premature chromosome condensation (PCC) technique was used to study several factors that determine the yield of chromosome fragments as observed in interphase cells after irradiation. In addition to absorbed dose and the extent of chromosome condensation at the time of irradiation, changes in chromosome conformation as cells progressed through the cell cycle after irradiation affected dramatically the yield of chromosome fragments observed. As a test of the effect of chromosome decondensation, irradiated metaphase Chinese hamster ovary (CHO) cells were allowed to divide, and the prematurely condensed chromosomes in the daughter cells were analyzed in their G 1 phase. The yield of chromosome fragments increased as the daughter cells progressed toward S phase and chromosome decondensation occurred. When early G 1 CHO cells were irradiated and analyzed at later times in G 1 phase, an increase in chromosome fragmentation again followed the gradual increase in chromosome decondensation. As a test of the effect of chromosome condensation, G 0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. It can be concluded that changes in chromosome conformation interfered with rejoining processes. In contrast, resting chromosomes (as in G 0 lymphocytes irradiated before fusion) showed efficient rejoining. These results support the hypothesis that cytogenetic lesions become observable chromosome breaks when chromosome condensation or decondensation occurs during the cell cycle.

Even though the premature chromosome condensation (PCC) technique has been shown to be a powerful cytogenetic tool, it has not yet been applied for biological dosimetry purposes. An improved and simple methodology for polyethylene glycol-mediated cell fusion and PCC induction has given the opportunity to explore the use of peripheral blood mononuclear cell prematurely condensed chromosomes as a biological dosimeter of absorbed radiation dose. This new approach offers several advantages, and it is presented as an alternative to the conventional cytogenetic technique. First, the chromosomal damage may be visualized in about 2 hr from the moment a blood sample is available. Consequently, the chances for interphase cell death or chromosomal repair are decreased. Second, the amount of blood required is only 0.5 ml. Third, the PCC analysis of about 100 mononuclear blood cells shows the radiation damage even at low doses, whereas hundreds of lymphocyte metaphases must be analyzed when conventional cytogenetic techniques are applied. Fourth, the linear dose-response relationships obtained may be easily standardized. The methodology used diminishes the possibility of differential results from one laboratory to another by avoiding the variables introduced by the stimulation and culture of lymphocytes required by conventional cytogenetic techniques. Finally, the kinetics of lymphocyte chromosome aberration recovery can be directly examined. This approach provides us, therefore, with a direct and more sensitive method for the estimation of radiation injury and repair.