Identification of Mre11 as a target for heat radiosensitization.

Thermal radiosensitization is believed to be mediated by an inhibition of double-strand break (DSB) repair, but the exact mechanism of radiosensitization remains to be elucidated. Previously, we demonstrated that proteins of the Mre11/Rad50/Nbs1 complex (MRN) translocate from the nucleus to the cytoplasm in cells have that been heated or heated and then irradiated; this finding led us to propose that heat radiosensitization was due at least in part to translocation of MRN. In the current study, we used leptomycin B to inhibit MRN translocation in heated, irradiated cells, but we found that heat radiosensitization was not altered. Thus enhanced radiosensitivity was not attributed to translocation of MRN proteins. To determine which of the MRN subunits contributed to heat radiosensitization, we compared the extent of heat radiosensitization in wild-type cells with that of cells hypomorphic for Mre11 or Nbs1 or cells in which the level of Rad50 was suppressed. We found that neither Nbs1 nor Rad50 is involved in heat radiosensitization, because a similar amount of heat radiosensitization was observed in cells deficient in those proteins compared to cells expressing normal levels. However, heat radiosensitization was not observed in A-TLD1 cells deficient in Mre11. Measurement of exonuclease activity of purified Mre11 heated at 42.5°C or 45.5°C indicated that the protein is very heat-labile. Immunoprecipitation of Mre11 from heated HeLa cells also revealed that hsp70 associates with Mre11 and that this association is maintained long after heating. Taken together, these findings implicate Mre11 as a target for heat radiosensitization and suggest that heat radiosensitization and inhibition of DSB repair may be mediated by heat-induced conformational changes in Mre11.

Age and hormonal status as determinants of cataractogenesis induced by ionizing radiation. I. Densely ionizing (high-LET) radiation.

Astronauts participating in extended lunar missions or the projected mission to Mars would likely be exposed to significant doses of high-linear energy transfer (LET) heavy energetic charged (HZE) particles. Exposure to even relatively low doses of such space radiation may result in a reduced latent period for and an increased incidence of lens opacification. However, the determinants of cataractogenesis induced by densely ionizing radiation have not been clearly elucidated. In the current study, we show that age at the time of exposure is a key determinant of cataractogenesis in rats whose eyes have been exposed to 2 Gy of (56)Fe ions. The rate of progression of cataractogenesis was significantly greater in the irradiated eyes of 1-year-old rats compared to young (56-day-old) rats. Furthermore, older ovariectomized rats that received exogenous estrogen treatment (17-ß-estradiol) commencing 1 week prior to irradiation and continuing throughout the period of observation of up to approximately 600 days after irradiation showed an increased incidence of cataracts and faster progression of opacification compared to intact rats with endogenous estrogen or ovariectomized rats. The same potentiating effect (higher incidence, reduced latent period) was observed for irradiated eyes of young rats. Modulation of estrogen status in the 1-year-old animals (e.g., removal of estrogen by ovariectomy or continuous exposure to estrogen) did not increase the latent period or reduce the incidence to that of intact 56-day-old rats. Since the rapid onset and progression of cataracts in 1-year-old compared to 56-day-old rats was independent of estrogen status, we conclude that estrogen cannot account for the age-dependent differences in cataractogenesis induced by high-LET radiation.

Effects of estrogen and gender on cataractogenesis induced by high-LET radiation.

Planning for long-duration manned lunar and interplanetary missions requires an understanding of radiation-induced cataractogenesis. Previously, it was demonstrated that low-linear energy transfer (LET) irradiation with 10 Gy of (60)Co gamma rays resulted in an increased incidence of cataracts in male rats compared to female rats. This gender difference was not due to differences in estrogen, since male rats treated with the major secreted estrogen 17-beta-estradiol (E2) showed an identical increase compared to untreated males. We now compare the incidence and rate of progression of cataracts induced by high-LET radiation in male and female Sprague-Dawley rats. Rats received a single dose of 1 Gy of 600 MeV (56)Fe ions. Lens opacification was measured at 2-4-week intervals with a slit lamp. The incidence and rate of progression of radiation-induced cataracts was significantly increased in the animals in which estrogen was available from endogenous or exogenous sources. Male rats with E2 capsules implanted had significantly higher rates of progression compared to male rats with empty capsules implanted (P = 0.025) but not compared to the intact female rats. These results contrast with data obtained after low-LET irradiation and suggest the possibility that the different types of damage caused by high- and low-LET radiation may be influenced differentially by steroid sex hormones.

Interstrain differences in the development of pyometra after estrogen treatment of rats.

This case report describes the unanticipated development of pyometra in Brown Norway rats after treatment with estrogen. Sprague Dawley and Brown Norway rats were ovariectomized and randomly assigned to treatment groups (subcutaneous implantation of either a capsule containing 20 mg 17beta-estradiol or an empty capsule, as a control). After irradiation of only the right eye, the rats were followed for several months in an attempt to determine the effects of estrogen on radiation cataractogenesis and investigate potential strain differences in this phenomenon. However, all Brown Norway rats that received estradiol treatment developed pyometra, whereas none the Sprague Dawley or control Brown Norway rats did. This case demonstrates the potential adverse effects of exogenous estrogen therapy, which are strain-specific in the rat. Caution should be taken when designing estrogen-related experiments involving Brown Norway rats and other potentially sensitive strains.

Effect of gender on radiation-induced cataractogenesis.

Radiation cataractogenesis is an important consideration for radiotherapy patients and for astronauts. Data in the literature suggest that gender and/or estrogen may play a role in the incidence of age-related cataracts. However, few data exist on the effect of gender on radiation-induced cataractogenesis. We compared the incidence and rate of progression of cataracts induced by ionizing radiation in male and female Sprague-Dawley rats. Male rats were implanted with either an empty silastic capsule or a capsule containing 17-beta-estradiol. Ovary-intact female rats were implanted with empty capsules. All rats received a single dose of 10 Gy (60Co gamma rays) to the right eye only. Lens opacification was measured at 2-4-week intervals with a slit lamp. The incidence of radiation-induced cataracts was significantly increased in male rats compared to female rats (P=0.034). There was no difference in the rate of cataract progression between the three groups. Our data suggest there is a gender-related difference in radiation-induced cataractogenesis, but the increased incidence of radiation cataractogenesis in male rats compared to female rats cannot be attributed to estrogen levels, since there was no difference in cataract incidence between male rats implanted with empty capsules and those implanted with capsules containing 17-beta-estradiol.