Makoto Ikebuchi

The enhancement of cell lethality and changes in production and repair of DNA damage by hypertonic treatment after exposure to X rays, bleomycin, and neocarzinostatin.

Chinese hamster ovary cells were treated with hypertonic 0.5 M NaCl solution after exposure to X rays or the radiomimetic drugs bleomycin or neocarzinostatin. The cytotoxicity of these agents was greatly enhanced by the hypertonic treatment. On the other hand, no effect was observed after exposure to ultraviolet light, and a significant effect was obtained with mitomycin C (MMC), adriamycin (ADR), and ethyl methanesulfonate (EMS). Assays by filter elution revealed that hypertonicity had various effects on the damage produced by the different agents. Strand breaks resulting from exposure to X rays and radiomimetic agents were sensitive to hypertonic treatment. Hypertonicity caused the production of new lesions and inhibited the rejoining of DNA strand breaks, both of which may be responsible for the enhanced cytotoxicity. On the other hand, the formation of crosslinks by MMC or protein-associated double-strand breaks by ADR, the major forms of damage by which these agents cause cytotoxicity, was not affected by hypertonic treatment. As strand breaks are known to be produced by MMC or ADR, they could account at least partly for the sensitization. However, various kinds of damage are produced by MMC, and any of these could be involved in the sensitization. To our knowledge EMS produces only base damage. Thus hypertonic treatment may have an effect on various types of damage.

Recovery from sublethal damage is reversibly inhibited by hypertonic saline: the effects of 0.23 M sodium chloride.

We previously showed that 0.23 M NaCl was able to fix slowly repairing potentially lethal damage (PLD), and that this slowly repairing PLD is distinct from rapidly repairing PLD that is sensitive to 0.5 M NaCl (Ikebuchi et al., Radiat. Res. 141, 19-27, 1995). In the present study, the effect of 0.23 M NaCl on repair of sublethal damage (SLD) was examined in cells of three rodent cell lines with normal radiosensitivity (Chinese hamster V79, BALB/c 3T3, RD13B2) and two radiosensitive lines derived from severe combined immunodeficient (scid) mice. Repair of SLD was detected as an increase in survival when the radiation dose was fractionated with an interval of incubation between the two doses. Repair of SLD occurred in V79 and BALB/c 3T3 cells but did not occur in the two scid cell lines which were defective in repair of double-strand breaks (DSBs), demonstrating that repair of DSBs is involved in repair of SLD. This was confirmed by the observation that repair of SLD occurred in RD13B2 cells, the scid line which had regained the ability to repair DSBs. When the V79 and BALB/c 3T3 cells were treated with 0.23 M NaCl during the interval between the split doses, repair of SLD was completely inhibited. On the other hand, repair of SLD occurred when the cells were incubated in culture medium between the treatment with 0.23 M NaCl and the second dose. From these observations, it is concluded that the inhibition of repair of SLD by 0.23 M NaCl is reversible, which is in contrast to the irreversible inhibition of repair of PLD by 0.23 M NaCl found previously. In addition, the fact that scid cells that were shown to have the ability to repair PLD that is sensitive to 0.23 M NaCl had little capacity to repair SLD suggests that there may be different processes involved in the two types of cellular repair.

Early changes in bone mineral content caused by acute irradiation are not observed after daily fractionation: A study by dual-energy X-ray absorptiometry

To reveal the effects of irradiation on bone metabolism in terms of time course and the effects of fractionation, early changes in bone mineral content (BMC) in rat tibiae were examined directly after irradiation. The left hind limbs of retired Wistar rats were exposed to a total dose of 60 Gy X-rays either acutely or 2.5 Gy daily fractionately. At 1–45 days (acutely irradiated) or 1–168 days (fractionately irradiated) later, the tibiae were removed and BMC was measured by dual-energy X-ray absorptiometry (DXA). On the computer screen tibiae were divided into four equal parts and the BMC in each was measured. Results were expressed as ratios of BMC in irradiated to nonirradiated tibiae for all parts. At 1–11 days after acute irradiation, changes in BMC ratios were not statistically significant (1.00±0.01 to 1.03±0.01,P>.05, respectively) in the upper part of the tibiae. At 16 days the ratio was 1.09±0.01 (P=.0001) and became 1.12±0.01 (P <.0001) and 1.16±0.02 (P<.0001) 22 and 45 days after irradiation, respectively. There were no statistically significant changes in BMC ratios in the remaining parts of the tibiae. Fractionated irradiation clearly resulted in no significant changes in BMC ratios. The threshold dose to initiate an increase in BMC on the 22nd day after acute irradiation was 20 Gy. These data suggest that early increase in mineral content, which is caused by a single large-dose of irradiation, can be delayed by daily fractionations.