Radiation-induced lethality and mutation in a repair-deficient CHO cell line.

A U.V.-sensitive, DNA repair-deficient mutant of Chinese hamster ovary cells was tested for its response to the lethal effects of X-irradiation and simulated solar light, and to the mutagenic actions of X-rays. A slight sensitivity to killing by X-rays and a greater sensitivity to solar light was observed relative to the wild-type CHO cells. More mutations were induced at a given dose of X-rays in the sensitive cell line than in the wild-type. These results are interpreted in terms of overlap in the repair processes which take place after U.V. damage in mammalian cells with those that take place after other types of radiation damage.

Repair capability and the cellular age response for killing and mutation induction after UV.

The cell-cycle response for killing and mutation induction by ultraviolet irradiation was measured in synchronous Chinese hamster ovary cells (CHO wild-type) and in a UV-hypersensitive mutant (43-3B) derived from this line. The CHO 43-3B line shows a greatly enhanced sensitivity to killing (D0 of 0.3 as compared to 3.2 J/m2 for the wild-type), is hypermutable, and deficient in DNA repair. For the wild-type, a characteristic age response is seen for killing by UV, with maximum sensitivity in early-S and resistance increasing through the S-phase. There is also a life-cycle specificity for induction of diphtheria-toxin resistance in late-G1 and early-S. Relatively little variation is seen through the cell cycle for induced 6-thioguanine and ouabain resistance. In contrast, the 43-3B cell line shows a relatively 'flat' response to UV throughout the cell cycle, for both killing and mutation induction. Therefore it appears that the characteristic age responses seen in the wild-type CHO are associated with the function of an essentially error-free repair process. A variation in the ability of this repair process to act in eliminating potentially lethal and mutagenic lesions (either due to a variation in repair enzyme activities through the cell cycle, or in the time available for repair) would account for most of the age response which is seen in the wild-type for killing and mutation induction by ultraviolet light.

UV-light-induced mutations in synchronous CHO cells.

Asynchronous and synchronous CHO cells were irradiated with germicidal UV light to determine the fluence response curve for cell killing, and the induction of resistance to 6-thioguanine, ouabain, and diphtheria toxin. For asynchronous populations the data show a sigmoidal response for induced reproductive death, as has been seen by others, with a D0 of 6 J/m2 and an extrapolation number of 2.5. The induction of mutations appears to be a linear function for all three mutagenic markers up to a dose of 17 J/m2. Reproductive death induced in the synchronous populations is a function of the time at which exposure occurs in the cell cycle, with late G1 and early S being the sensitive stages. The induction of resistance to 6TG, ouabain, and diphtheria toxin (DT) all seem to depend on the time of exposure in the cell cycle. As in the case of UV-induced reproductive death, the more sensitive periods for mutation induction appear also to be the G1 and early S period of the cell cycle, with the largest cyclic variation occurring for induced DT resistance. A comparison of the results reported here for the UV exposure with exposures of synchronous CHO cells to X-rays and ethylnitrosourea suggests that there are different age-specific responses to mutation induction for each agent, and that there are often different age responses for different mutagenic endpoints with the same mutagen.

Ethylnitrosourea-induced mutagenesis in asynchronous and synchronous Chinese hamster ovary cells.

The toxic and mutagenic activity of the alkylating carcinogen N-ethyl-N-nitrosourea (ENU) was studied in Chinese hamster ovary (CHO) cells. Cell killing and induction of 6-thioguanine-resistant (TGr) and ouabain-resistant (OUAr) mutants were determined as a function of ENU dose and treatment time in asynchronous cell populations. A dose-dependent induction of mutants was observed. The mutation frequency did not increase with longer than 30-min treatment times, implying that ENU breaks down rapidly in the cell. When synchronous populations of CHO cells obtained by mitotic detachment were treated with ENU at various times during the cell cycle, ENU-induced reproductive death was strongly dependent on the position in the cell cycle at the time of treatment, the time of highest sensitivity being the beginning of the S period. The pattern of mutation induction by ENU over the cell cycle was quite different from the pattern for cell killing. The induction of TGr mutants seemed to be independent of cell-cycle time. The induction of OUAr mutants was also independent of cell-cycle time after a low ENU dose; however, after a high ENU dose the frequency of OUAr mutants varied during the cell cycle, with a slight enhancement in B1 and a decrease in the early S period. There was no sign of enhanced mutation induction at the growing point for the two genetic markers tested.

Bromodeoxyuridine-induced mutations in synchronous Chinese hamster cells: temporal induction of 6-thioguanine and ouabain resistance during DNA replication.

Mutations were induced in synchronous Chinese hamster cells by bromodeoxyuridine (BUdR) incorporated into cells for one-hour periods in the cell cycle. There was a very pronounced temporal dependence during the first half of the DNA synthesis period for the induction of damage leading to 6-thioguanine (6TG) and ouabain resistance. No mutants above background were induced by exposure to BUdR in G1 and G2 cells, and very few mutants were induced in the latter part of the DNA synthesis period. The peak for the induction of 6TG resistance occurs at about two hr in the DNA synthesis period; one hour later there is a peak for the induction of ouabain resistance. Both peaks occur before the time of maximum incorporation of BUdR into DNA. These results suggest that the mutagenesis by BUdR is associated with at least two nuclear genes, which replicate at two hr and three hr in the DNA synthesis period.

Molecular suicide studies of 125I and 3H disintegration in the DNA of Chinese hamster cells.

The shape of the survival curve for cells inactivated by tritium decay in DNA is modified by the presence of halogenated pyrimidines in the DNA in a manner analogous to their effect on X-ray induced reproductive death. The large shoulder found for tritium suicide is removed completely by coincorporation of 10(-6) M IUdR. The oxygen enhancement ratio for 125I and tritium disintegrations in unsynchronized Chinese hamster cells was determined for cells permitted to accumulate damage from these events at 4 degrees C. The oxygen enhancement ratio for 125I induced damage is 1.4. This is much smaller than the OER found for tritium decay which is similar or more than that found for X-ray exposure under the same conditions. These results suggest that the nature of the lesions produced by 125I decay in DNA are analogous to those produced by high LET radiation while those lesions produced by tritium are similar to lesions produced by roentgen rays. In synchronous V79 cells the effects of 125I induced damage in different regions of the mammalian cell DNA was examined taking advantage of the fact that DNA replication in hamster nuclei follows a time-dependent three dimensional pattern. The experiments indicate that 125I decays accumulated in the G2-period of the cell cycle have different efficiences for the induction of reproductive death depending on the region of the DNA which is labeled. The efficiency for the induction of reproductive death appears to be a maximum in DNA that replicates in V79 cells near the end of the DNA replication cycle. Electron capture events are dramatically efficient in the production of lethal chromosome aberrations. In CHO cells synchronized in the G1-stage of the cell cycle stored in the frozen state the efficiency for the induction of dicentric and ring chromosomes is 0.03. The dose response curve for the induction of these aberrations is linear in contrast to the curvilinear response found for roentgen ray exposure under the same conditions. Data on this kind suggest that there may exist "critical" regions within mammalian cell nuclei where chromatin fibers from two different chromosomes are in close proximity to each other and both are damaged non-repairably by a single electron capture event.

The oxygen-enhancement ratio for reproductive death induced by 3H or 125I damage in mammalian cells.

The oxygen-enhancement ratio (o.e.r.) for 3H- and 125I-induced cell death at 4 degrees C was determined in cultured Chinese hamster cells. The o.e.r. for cell death induced by 3H-thymidine was 3-2, essentially the same value as that previously reported for X-ray induced cell death. For cell death induced by 125I-iododeoxyuridine (125IdUrd), the o.e.r. was less than 1-4. The lower o.e.r. for 125I-induced death was not due to the presence of the base analogue itself, since cells that had incorporated unlabelled IdUrd and were X-irradiated had an o.e.r. of 2-8 and cells that were inactivated by 3H-IdUrd damage at 4 degrees C had an o.e.r. even greater than 3. These results suggest that 125I-decay damage, like high-linear-energy-transfer radiation damage, is only slightly increased by the presence of oxygen.

Survival of synchronized Chinese hamster cells exposed to radiation of different linear-energy transfer.

Chinese hamster V79 cells were exposed to ionizing radiations of a wide range of linear-energy transfer (LET), including 145kV x-rays and six different heavy ions accelerated in the Berkeley heavy-ion linear accelerator. The LET of the ions ranged from 19 keV/um to 2000 keV/um. Survival curves were determined for both synchronized and asynchronous cells, using survival of colony-forming capacity as the end-point. Results with asynchronous cultures were similar to results reported previously for mammalian cells. There was increased effectiveness of killing per dose with increased LET until a change in shape of the single-cell survival curves resulted, from sigmoidal to exponential, with carbon ions (LET of 190 keV/um). With heavier ions, exponential curves were obtained, but with decreased effectiveness per unit dose. Synchronized cultures were obtained by mitotic selection. The expected Chinese hamster cell-cycle survival curve variation was found for X-rays, mainly reflecting the variation in the single-cell extrapolation number, with late S-phase cells the most resistant to radiation. When synchronized cultures were irradiated with the heavy ions that produce exponential survival curves; the survival curves were independent of the cell-cycle time of irradiation. With radiations of LET values between the low and high extremes, a reduced cell-cycle survival curve variation was found, indicating a gradual reduction in the cell-cycle survival curve variation as a function of increased LET.