high acute doses of ionizing radiation are known to be harmful to living organisms . however , at low doses and dose rates , radioadaptive responses have been observed in vitro and in vivo using various end points , such as cell lethality , chromosomal aberrations , mutation induction , radiosensitivity , and dna repair . adaptation in vitro is most efficiently induced by doses of 1 to 50 cgy at dose rates from 0.6 to 60 gy / h , with challenge doses in the range of 0.5 to 2 gy . the protective effect has been reported to last for up to 3 cell generations following acute priming irradiation in lymphocytes . 
  in vivo an adaptive effect of an acute priming dose of 2.5 to 50 cgy has been seen in measurements of 30-day survival of icr mice after a challenge dose of 8 gy . other types of adaptive or improved responses have been measured in vivo after low - dose - rate ( ldr ) priming irradiation ; long - term ldr preirradiation has been shown to reduce the incidence of chemically and radiation - induced tumors . several studies have shown prolongation of life span after long - term ldr irradiation for mice not given a challenge dose . one should , however , notice that there are also studies that contrast to these findings . for example , a study by shin et al showed that the average life span was significantly lower than unirradiated controls in akr / j mice irradiated with a total of 4.5 gy at either ldr ( 0.7 mgy / h ) or high dose rate ( 48 gy / h ) . however , the incidence of thymic lymphoma was lower in ldr irradiated mice ( ie , 10% lower than control ; 20% lower than high dose rate ) . significant life shortening was also observed by thomson et al in male b6cf1 mice ( a mouse strain in which mortality is affected by the incidence of various cancers ) exposed to 0.8 mgy / h for 23 weeks or 7.5 mgy / h for 59 weeks and by tanaka et al using the same mouse strain irradiated for approximately 400 consecutive days ( starting week 8) with a dose rate of 21 mgy / d . tanaka et al however found no effect on life span for dose rates of 1.1 or 0.05 mgy / d . other studies found no effect on life span by chronic irradiation at dose rates of 1 and 2 mgy / h , 4 mgy / h and 50 cgy / h , or 10 cgy / year ( 0.011 mgy / h ) . 
  in the present study , we have given dba/2 mice an ldr whole - body priming dose of 30 cgy given over 1 hour ( 30 cgy / h ) . the priming irradiation used in the present study was chosen on the basis of our earlier studies partly performed on in vitro cells and partly on dba/2 mice . the background for that was our earlier finding that 30 cgy given acutely to human t-47d cells induced cell death in about 20% of the cells ( hyperradiosensitivity ) , whereas the same dose given protracted during 1 hour ( 30 cgy / h ) did not induce significant cell killing . our data furthermore indicated that transforming growth factor 3 ( tgf-3 ) was induced by this priming irradiation and that addition of active tgf-3 to the cells before irradiation could even protect t-47d cells against a larger challenge dose given after the priming dose . 
  in the follow - up to these in vitro studies , we used the t-47d cells as in vitro reporter cells to test whether these mechanisms could be initiated by priming irradiation of an animal : we gave dba/2 mice a whole - body priming dose of 30 cgy at the ldr of 30 cgy / h and subsequently harvested serum , which was transferred to reporter t-47d cells cultured in vitro . the response of the reporter cells to a challenge dose in the presence of the mouse serum was then tested . it turned out that serum from unprimed mice had no effect on the radiosensitivity of the reporter cells , while serum from primed mice increased radioresistance of the reporter cells to all doses given ( up to 5 gy was tested ) . the effect of the serum was seen to depend on tgf-3 during reporter cell exposure and on inducible nitric oxide synthase ( inos ) activity in the mice before the serum was harvested . 
  in the light of these results , we wondered if the mechanisms activated in the mouse body by the priming dose could not only protect reporter cells against a challenge dose but perhaps even improve the mouse survival after a whole - body , lethal challenge dose ( ld50 ) . we conclude that whole - body preirradiation with 30 cgy / h protects dba/2 mice against a subsequent single whole - body ld50 ( 9 - 9.5 gy ) challenge dose , but it does not affect the life span of mice not given a challenge dose . inbred nonspecific pathogen - free dba/2 mice were used . after sex segregation at weaning , the animal room , the animal care , and the experimental use of the animals were in accordance with the slovak ethical rules . the mice were maintained , cared , and used in accordance with institutional guidelines under the approved protocols by the ethical committee and the slovak veterinary office . mice were collected for each experiment as they were bred with the result that the age at the time of the priming irradiation could vary from 2 to 5 months . animals were placed in a circular pen with wedge - shaped individual rooms , giving each animal an identical exposure to the radiation . a co - source ( theratron elite 100 ; best theratronics , canada ) the priming dose rate was obtained with total cerrobend filtration in solid poly(methyl methacrylate ) [ pmma ] phantom by optimal source to skin distance ( ssd ) ( 217 cm ) for a dose rate of the 30 cgy / h with field size 35  35 cm . the time for application of 30 cgy was in the interval 59.6 to 60.3 minutes . the treatment parameters were corrected at each application to ensure that the mice received the demanded dose rate . the dose rate was controlled by an ionization chamber ( fc-65 g ; wellhfer , germany ) calibrated by standard laboratory iaea seibersdorf . two different types of experiments were conducted . in the first 2 experiments ( figures 1 and 2 ) , primed and nonprimed mice were exposed to a challenge dose of 9 or 9.5 gy . sixty - four mice ( 16 females and 16 males were primed and the same number nonprimed ) were used in the experiment shown in figure 1 , and 36 mice ( 12 females and 8 males were primed and 8 of each nonprimed ) were used in the experiment shown in figure 2 . the dose rate used for challenge doses of 9 and 9.5 gy was 55.8 gy / h , and the time between priming and challenge irradiation was about 6 weeks . in order to minimize the possible effects of age - related differences in responses to the priming irradiation , sixteen males and 16 females were first given a 1-hour priming irradiation at 30 cgy / h . six weeks later , they were irradiated with a high - dose - rate challenge dose of 9 gy . sixteen males and 16 females ( age matched to the primed mice ) were challenge irradiated without a priming dose . eight males and 12 females were first given a 1-hour priming irradiation at 30 cgy / h , 6 weeks before the high - dose - rate challenge dose of 9.5 gy . eight males and 8 females ( age matched to the primed mice ) were challenge irradiated without a priming dose . ninety - nine mice ( 47 females and 52 males ) were primed with 30 cgy for 1 hour , and 159 ( 47 females and 112 males ) were used as controls . the mice were monitored regularly every second day by estimation of their weight . in the period of frequent death because of irradiation , they were checked every day . mice with life expectancy of few hours ( moribund ) were euthanized by an overdose of anesthetics . life span in mice exposed to 1-hour priming irradiation at 30 cgy / h compared to unprimed mice . the effect of age at the time of priming on life span is negligible ( shown in supplement figure 3 ) . a , males and females pooled . the mice in the life span study ( figure 3 ) died of old age after turning gray . in the mice irradiated with the challenge doses ( figures 1 and 2 ) , a gradual loss in body weight was observed at first . at autopsy , diarrhea or hair loss was not observed , but some of the mice turned gray . statistical analyses were performed using student t test and mantel - cox ( log rank ) test to compare life spans of ldr primed mice versus unprimed mice ( figure 3 ) . for the data where a challenge radiation dose of either 9 or 9.5 gy was given with or without previous priming ( figures 1 and 2 ) , mantel - cox ( log rank ) test was used . inbred nonspecific pathogen - free dba/2 mice were used . after sex segregation at weaning , the animal room , the animal care , and the experimental use of the animals were in accordance with the slovak ethical rules . the mice were maintained , cared , and used in accordance with institutional guidelines under the approved protocols by the ethical committee and the slovak veterinary office . mice were collected for each experiment as they were bred with the result that the age at the time of the priming irradiation could vary from 2 to 5 months . animals were placed in a circular pen with wedge - shaped individual rooms , giving each animal an identical exposure to the radiation . a co - source ( theratron elite 100 ; best theratronics , canada ) the priming dose rate was obtained with total cerrobend filtration in solid poly(methyl methacrylate ) [ pmma ] phantom by optimal source to skin distance ( ssd ) ( 217 cm ) for a dose rate of the 30 cgy / h with field size 35  35 cm . the time for application of 30 cgy was in the interval 59.6 to 60.3 minutes . the treatment parameters were corrected at each application to ensure that the mice received the demanded dose rate . the dose rate was controlled by an ionization chamber ( fc-65 g ; wellhfer , germany ) calibrated by standard laboratory iaea seibersdorf . two different types of experiments were conducted . in the first 2 experiments ( figures 1 and 2 ) , primed and nonprimed mice were exposed to a challenge dose of 9 or 9.5 gy . sixty - four mice ( 16 females and 16 males were primed and the same number nonprimed ) were used in the experiment shown in figure 1 , and 36 mice ( 12 females and 8 males were primed and 8 of each nonprimed ) were used in the experiment shown in figure 2 . the dose rate used for challenge doses of 9 and 9.5 gy was 55.8 gy / h , and the time between priming and challenge irradiation was about 6 weeks . in order to minimize the possible effects of age - related differences in responses to the priming irradiation , sixteen males and 16 females were first given a 1-hour priming irradiation at 30 cgy / h . six weeks later , they were irradiated with a high - dose - rate challenge dose of 9 gy . sixteen males and 16 females ( age matched to the primed mice ) were challenge irradiated without a priming dose . eight males and 12 females were first given a 1-hour priming irradiation at 30 cgy / h , 6 weeks before the high - dose - rate challenge dose of 9.5 gy . eight males and 8 females ( age matched to the primed mice ) were challenge irradiated without a priming dose . ninety - nine mice ( 47 females and 52 males ) were primed with 30 cgy for 1 hour , and 159 ( 47 females and 112 males ) were used as controls . the mice were monitored regularly every second day by estimation of their weight . in the period of frequent death because of irradiation , they were checked every day . mice with life expectancy of few hours ( moribund ) were euthanized by an overdose of anesthetics . life span in mice exposed to 1-hour priming irradiation at 30 cgy / h compared to unprimed mice . the effect of age at the time of priming on life span is negligible ( shown in supplement figure 3 ) . a , males and females pooled . the mice in the life span study ( figure 3 ) died of old age after turning gray . in the mice irradiated with the challenge doses ( figures 1 and 2 ) , a gradual loss in body weight was observed at first . at autopsy , diarrhea or hair loss was not observed , but some of the mice turned gray . statistical analyses were performed using student t test and mantel - cox ( log rank ) test to compare life spans of ldr primed mice versus unprimed mice ( figure 3 ) . for the data where a challenge radiation dose of either 9 or 9.5 gy was given with or without previous priming ( figures 1 and 2 ) , mantel - cox ( log rank ) test was used . figure 1 shows the fraction of mice surviving as a function of the time after a 9 gy whole - body challenge dose . separate curves are plotted for animals given a prechallenge priming dose of 0.3 gy with an ldr of 0.3 gy / h 6 weeks before the challenge dose and for animals given the challenge dose without any priming irradiation . the pooled data ( figure 1a ) indicate that the median survival time increased from 90 days for unprimed mice to over 230 days for primed mice . this difference is not statistically significant by a mantel - cox ( log rank ) test ( p = .08 ) . there is , however , a marked difference between the genders : a significant increase in life span in primed compared to unprimed mice was seen in females ( figure 1b ) . the data on males however indicated only a slight increase in the life span for the primed compared to the unprimed animals , a difference not statistically significant ( figure 1c ) . the experiment was repeated with a slightly higher challenge dose of 9.5 gy but with the same priming irradiation as was used in the first experiment ( figure 2 ) . unfortunately , the follow - up time in the second experiment had to be shortened to just 81 days due to technical reorganization of the animal house . in the cage , holding female mice given a priming dose , no deaths were observed during these 81 days of follow - up . in this second experiment , however , there was a significantly increased life span for primed versus unprimed mice when data for both genders were pooled together ( p = .001 ) . the statistical significance was also clear for both genders separately ( figure 2b , p = .02 for females and figure 2c , p = .001 for males ) . it is not known whether the protective effect of priming irradiation on a subsequent acute challenge irradiation has to do with increased repair capacity within individual cells or perhaps some systemic effect like , for example , stimulation of the immune system protecting the mice from detrimental effects from the microflora . such detrimental effects could also be of importance for the normal life span of the animals , that is , animals not exposed to any challenge dose , experiencing senescence before death . we therefore wanted to test whether the ldr priming had any protective or detrimental long - term effects in mice not given a challenge dose . a test of this kind , without a challenge dose , needs a high number of animals and a very long follow - up time . in our test , the life span of 99 ldr primed mice was compared to that of 159 unprimed controls with a follow - up of 1200 days , which roughly covers the normal life duration for a mouse ( figure 3 ) . the data showed no significant difference between the life spans of primed compared to unprimed mice . mantel - cox test gave p values of .66 for females , .11 for males , and .23 for pooled data from both genders . there was , however , a higher mortality rate in the earlier time intervals for the primed females than any of the other populations ( see supplementary figure s1 ) . in order to see if there could be specific effects related to a certain cage , we replotted the data from figure 3 also with individual time of death for each animal per cage in supplementary figure s2 . apart from an observation of 3 very long - lived unprimed females living in the same cage , there was no indication of special effects related to single cages . the mice were bred locally and collected for priming , which could only be done at certain times . the effect of age at the time of priming irradiation on the mean life span is plotted in supplementary figure s3 . linear fittings gave for primed females a slope of 1.5  0.9 , and for males 2.1  4.4 . thus , a correlation can not be ruled out for the females who also have the largest variation in age at priming . in the present study , the time between priming and challenge irradiation was 6 weeks . , we found that radioprotective effects were seen in reporter cells exposed to serum from primed mice even when the serum was harvested up to 15 months after priming irradiation . in both cases , involvement of tgf-3 and inos activity was demonstrated , which makes it tempting to speculate that tgf-3 and inos activity may play a role in the adaptive effect observed in figures 1 and 2 . it can therefore be debated whether the priming dose / dose rate should be adjusted for humans as compared to mice . our earlier in vitro studies showed that ldrs of 30 cgy / h as used in the present study ( 6 cgy / h was the lowest tested ) induced the same effect as long as the irradiation time was as prolonged as 1 hour . the protective effect vanished if the priming irradiation time was reduced to 15 minutes . in the experiments with reporter cells exposed to serum from irradiated mice , the effect seems thus to be dose independent as long as the dose rate is within a certain window and the irradiation time is long enough . our data confirm that the used priming dose induced similar mechanisms in mice as in human cell cultures . in light of the improved survival for the primed compared to the unprimed animals after a lethal challenge dose , it was interesting to evaluate whether the priming dose used had an impact on the life span of mice not exposed to a lethal irradiation dose . however , no significant differences were seen regarding life span in the primed versus unprimed mice ( figure 3a ) . there seems to be a slight tendency of longer life span in primed males compared to controls ( figure 3c ) , whereas the opposite trend appears for females ( figure 3b ) . the primed males also seemed to live longer ( not significant ) than primed females ( p = .16 ) . in the females , the data show some early deaths in the primed mice and 3 very long - lived controls . the early deaths in 2 cages coincided with early age at the time of priming ( supplementary figure s3 ) . interestingly , the 3 long - lived female controls lived in the same cage , and the early deaths in the primed females also seemed to appear in certain cages ( supplementary figure s2 ) . thus , it is possible that the life span in the females was influenced by unknown interactions between mice housed in the same cage and a larger variation in age at priming than in the males , in particular , 3 cages primed before 37 days of age . altogether , we conclude that with the size of the present study , it is not possible to see any significant change in life span after 1 hour of -irradiation at 30 cgy / h in otherwise unirradiated mice . the variations are more likely related to other factors in connection with keeping mice in cages of 8 in a nonbarrier unit and variations in age at the time of priming . the international commission on radiological protection suggests a threshold dose for damaging effects of 10 cgy . our previous and present data indicate that with an ldr of 30 gy / h , even a dose of 30 cgy to the mice results in a response characterized by no change in life span . this is in line with a study by thomson et al in which b6cf1 mice were irradiated with cobalt-60 -rays . the dose rates were adjusted to give 22.5 , 45 , or 90 cgy in 20 minutes , corresponding to 67.5 , 135 , and 270 cgy / h . the 2 lowest doses / dose rates are comparable to the irradiation used in the present study , and no significant change in life span was observed after these . 
  in conclusion , dba/2 mice given an ld50 of 9 or 9.5 gy experienced significantly improved long - term survival if they had been given a 1-hour whole - body priming -irradiation with 30 cgy before the lethal challenge dose as compared to mice without priming irradiation . the protection seems to be long lasting as the lethal challenge radiation doses were given 6 weeks after priming . no significant change in life span was observed due to priming in the mice not given a challenge dose .
prior findings showed that serum from dba/2 mice that had been given whole - body irradiation for 1 hour at a low dose rate ( ldr ) of 30 cgy / h induced protection against radiation in reporter cells by a mechanism depending on transforming growth factor 3 and inducible nitric oxide synthase activity . in the present study , 
 the effect of the 1 hour of ldr irradiation on the response of the preirradiated mice to a subsequent lethal dose and on the life span is examined . 
 these dba/2 mice were prime irradiated for 1 hour at 30 cgy / h . 
 two experiments with 9 and 9.5 gy challenge doses given 6 weeks after priming showed increased survival in primed mice compared to unprimed mice followed up to 225 and 81 days after challenge irradiation , respectively . 
 there was no overall significant difference in life span between primed and unprimed mice when no challenge irradiation was given . 
 the males seemed to have a slight increase in lifespan after priming while the opposite was seen for the females .