Influence of dose rate on the induction of simple and complex chromosome exchanges by gamma rays

Single-color painting of whole chromosomes, or protocols in which only a few chromosomes are distinctively painted, will always fail to detect a proportion of complex exchanges because they frequently produce pseudosimple painting patterns that are indistinguishable from those produced by bona fide simple exchanges. When 24-color multi-fluor FISH (mFISH) was employed for the purpose of distinguishing (truly) simple from pseudosimple exchanges, it was confirmed that the acute low-LET radiation dose-response relationship for simple exchanges lacked significant upward curvature. This result has been interpreted to indicate that the formation of simple exchanges requires only one chromosome locus be damaged (e.g. broken) by radiation to initiate an exchange-not two, as classical cytogenetic theory maintains. Because a one-lesion mechanism implies single-track action, it follows that the production of simple exchanges should not be influenced by changes in dose rate. To examine this prediction, we irradiated noncycling primary human fibroblasts with graded doses of (137)Cs gamma rays at an acute dose rate of 1.10 Gy/min and compared, using mFISH, the yield of simple exchanges to that observed after exposure to the same radiation delivered at a chronic dose rate of 0.08 cGy/min. The shape of the dose response was found to be quasi-linear for both dose rates, but, counter to providing support for a one-lesion mechanism, the yield of simple aberrations was greatly reduced by protracted exposure. Although chronic doses were delivered at rates low enough to produce damage exclusively by single-track action, this did not altogether eliminate the formation of complex aberrations, an analysis of which leads to the conclusion that a single track of low-LET radiation is capable of inducing complex exchanges requiring up to four proximate breaks for their formation. For acute exposures, the ratio of simple reciprocal translocations to simple dicentrics was near unity.     

Effects of tritiated water on germ cells in medaka embryos

Embryos of medaka, Oryzias latipes, were exposed to tritiated water and 137Cs gamma rays continuously from the one-cell stage until hatching (10 days at 26 degrees C). Germ cells in the gonads of newly hatched fry were counted in histological sections and compared with controls. The accumulated dose for 50% survival of germ cells was 195 rad for tritium beta rays and 350 rad for 137Cs gamma rays. Female progeny were produced using Yamamoto's method. The 50% survival doses for female germ cells treated in a manner similar to that described above were 140 rad for beta rays and 305 rad for gamma rays. When embryos of medaka were irradiated with gamma rays below an accumulated dose of 475 rad or treated with tritiated water at a concentration of 0.2 mCi/ml or lower, the dose response of the germ cells showed an exponential relationship. It appeared that there was no threshold or significant dose-rate effect for either beta or gamma rays on germ cell survival, and that tritium beta rays were more effective than 137Cs gamma rays in germ cell killing.     

The effectiveness of monoenergetic neutrons at 565 keV in producing dicentric chromosomes in human lymphocytes at low doses

The induction of dicentric chromosomes in human lymphocytes from one individual irradiated in vitro with monoenergetic neutrons at 565 keV was examined to provide additional data for an improved evaluation of neutrons with respect to radiation risk in radioprotection. The resulting linear dose-response relationship obtained (0.813 +/- 0.052 dicentrics per cell per gray) over the dose range of 0.0213-0.167 Gy is consistent with published results obtained for irradiation with neutrons from different sources and with different spectra at energies lower than 1000 keV. Comparing this value to previously published "average" dose-response curves obtained by different laboratories for (60)Co gamma rays and orthovoltage X rays resulted in maximum RBEs (RBE(m)) of about 37 +/- 8 and 16 +/- 4, respectively. However, when our neutron data were matched to low-LET dose responses that were constructed several years earlier for lymphocytes from the same individual, higher values of RBE(m) resulted: 76.0 +/- 29.5 for (60)Co gamma rays and 54.2 +/- 18.4 for (137)Cs gamma rays; differentially filtered 220 kV X rays produced values of RBE(m) between 20.3 +/- 2.0 or 37.0 +/- 7. 1. The results highlight the dependence of RBE(m) on the choice of low-LET reference radiation and raise the possibility that differential individual response to low-LET radiations may need to be examined more fully in this context.     

Cell death (apoptosis) in mouse intestine after continuous irradiation with gamma rays and with beta rays from tritiated water

Apoptosis is a pattern of cell death involving nuclear pycnosis, cytoplasmic condensation, and karyorrhexis. Apoptosis induced by continuous irradiation with gamma rays (externally given by a 137Cs source) or with beta rays (from tritiated water injected ip) was quantified in the crypts of two portions of mouse bowel, the small intestine and descending colon. The time-course change in the incidence of apoptosis after each type of radiation could be explained on the basis of the innate circadian rhythm of the cells susceptible to apoptotic death and of the excretion of tritiated water (HTO) from the body. For 6-h continuous gamma irradiation at various dose rates (0.6-480 mGy/h) and for 6 h after injection of HTO of various radioactivities (0.15-150 GBq per kg body wt), the relationships between dose and incidence of apoptosis were obtained. Survival curves were then constructed from the curves for dose vs incidence of apoptosis. For the calculation of the absorbed dose from HTO, the water content both of the mouse body and of the cells was assumed to be 70%. One megabecquerel of HTO per mouse (i.e., 40 MBq/kg body wt) gave a dose rate of 0.131 mGy/h. The mean lethal doses (D0) were calculated for gamma rays and HTO, and relative biological effectiveness values of HTO relative to gamma rays were obtained. The D0 values for continuous irradiation with gamma rays were 210 mGy for small intestine and 380 mGy for descending colon, and the respective values for HTO were 130 and 280 mGy, indicating the high radiosensitivity of target cells for apoptotic death. The relative biological effectiveness of HTO relative to 137Cs gamma rays for cell killing in both the small intestine and the descending colon in the mouse was 1.4-2.1.     

Induction of chromosome aberrations in G0 human lymphocytes by low doses of ionizing radiations of different quality

Human peripheral blood lymphocytes from two donors were exposed to low doses (0.05 to 2.0 Gy) of gamma rays, X rays, or fast neutrons of different energies. Chromosome aberrations were analyzed in metaphase of first-division cells after a culture time of 45-46 hr. At this time, less than 5% of the cells were found in second division. Different dose-response relationships were fitted to the data by using a maximum likelihood method; best fits for radiation-induced dicentric aberrations were obtained with the linear-quadratic law for all radiations. The linear component of this equation predominated, however, for neutrons in the range of doses studied, and the frequency of dicentrics induced by d(16)+Be neutrons up to 1.0 Gy could also be described by a linear relationship. The relative biological efficiency (RBE) of X rays and d(16)+Be, d(33)+Be, and d(50)+Be neutrons compared to 60Co gamma rays in the low dose range was calculated from the dose-effect relationships for the dicentrics produced. The RBE increased with decreasing neutron dose and with decreasing neutron energy from d(50)+Be to d(16)-+Be neutrons. The limiting RBE at low doses (RBEo) was calculated to be about 1.5 for X rays and 14.0, 6.2, and 4.7 for the d(16)+Be, d(33)+Be, and d(50)+Be neutrons, respectively.     

Induction of genomic instability in TK6 human lymphoblasts exposed to 137Cs gamma radiation: comparison to the induction by exposure to accelerated 56Fe particles

The induction of genomic instability in TK6 human lymphoblasts by exposure to (137)Cs gamma radiation was investigated by measuring the frequency and characteristics of unstable clones isolated approximately 36 generations after exposure. Clones surviving irradiation and control clones were analyzed for 17 characteristics including chromosomal aberrations, growth defects, alterations in response to a second irradiation, and mutant frequencies at the thymidine kinase and Na(+)/K(+) ATPase loci. Putative unstable clones were defined as those that exhibited a significant alteration in one or more characteristics compared to the controls. The frequency and characteristics of the unstable clones were compared in clones exposed to (137)Cs gamma rays or (56)Fe particles. The majority of the unstable clones isolated after exposure to either gamma rays or (56)Fe particles exhibited chromosomal instability. Alterations in growth characteristics, radiation response and mutant frequencies occurred much less often than cytogenetic alterations in these unstable clones. The frequency and complexity of the unstable clones were greater after exposure to (56)Fe particles than to gamma rays. Unstable clones that survived 36 generations after exposure to gamma rays exhibited increases in the incidence of dicentric chromosomes but not of chromatid breaks, whereas unstable clones that survived 36 generations after exposure to (56)Fe particles exhibited increases in both chromatid and chromosome aberrations.     

Effect of dose rate on the survival of irradiated human skin fibroblasts.

The survival of cells in density-inhibited, confluent cultures maintained at 37 degrees C was examined following exposure to 137Cs gamma rays at low dose rates (0.023 or 0.153 Gy/h) or to 60Co gamma rays at a single high dose rate (0.70-0.75 Gy/min). Cells from an ataxia telangiectasia (AT) homozygote showed no dose-rate effect, whereas a three- to fivefold increase in D0 was observed for all other cell strains exposed at low dose rates. The magnitude of the dose-rate effect did not differ significantly among cells from persons with hereditary retinoblastoma, basal cell nevus syndrome, or AT-heterozygote compared with normal cell strains, and was not related to the size of the shoulder (extrapolation number) of the survival curve. Furthermore, no differences in the capacity for the repair of potentially lethal damage during confluent holding were observed among these latter cell strains.     

No lengthening of life span in mice continuously exposed to gamma rays at very low dose rates

Late effects of continuous exposure to ionizing radiation are potential hazards to workers in radiation facilities as well as to the general public. Recently, low-dose-rate and low-dose effects have become a serious concern. Using a total of 4000 mice, we studied the late biological effects of chronic exposure to low-dose-rate radiation as assayed by life span. Two thousand male and 2000 female 8-week-old specific-pathogen-free (SPF) B6C3F1 mice were randomly divided into four groups (one nonirradiated control and three irradiated). Irradiation was carried out for approximately 400 days using (137)Cs gamma rays at dose rates of 21 mGy day(-1), 1.1 mGy day(-1) and 0.05 mGy day(-1) with total doses equivalent to 8000 mGy, 400 mGy and 20 mGy, respectively. All mice were kept under SPF conditions until they died spontaneously. Statistical analyses showed that the life spans of mice of both sexes irradiated with 21 mGy day(-1) (P < 0.0001) and of females irradiated with 1.1 mGy day(-1) (P < 0.05) were significantly shorter than those of the control group. Our results show no evidence of lengthened life span in mice continuously exposed to very low dose rates of gamma rays.     

Measurement of the initial levels of DNA damage in human lymphocytes induced by 29 kV X rays (mammography X rays) relative to 220 kV X rays and gamma rays

Experiments using the alkaline comet assay, which measures all single-strand breaks regardless of their origin, were performed to evaluate the biological effectiveness of photons with different energies in causing these breaks. The aim was to measure human lymphocytes directly for DNA damage and subsequent repair kinetics induced by mammography 29 kV X rays relative to 220 kV X rays, 137Cs gamma rays and 60Co gamma rays. The level of DNA damage, predominantly due to single-strand breaks, was computed as the Olive tail moment or percentage DNA in the tail for different air kerma doses (0.5, 0.75, 1, 1.5, 2 and 3 Gy). Fifty cells were analyzed per slide with a semiautomatic imaging system. Data from five independent experiments were transformed to natural logarithms and fitted using a multiple linear regression analysis. Irradiations with the different photon energies were performed simultaneously for each experiment to minimize interexperimental variation. Blood from only one male and one female was used. The interexperimental variation and the influence of donor gender were negligible. In addition, repair kinetics and residual DNA damage after exposure to a dose of 3 Gy were evaluated in three independent experiments for different repair times (10, 20, 30 and 60 min). Data for the fraction of remaining damage were fitted to the simple function F(d) = A/(t + A), where F(d) is the fraction of remaining damage, t is the time allowed for repair, and A (the only fit parameter) is the repair half-time. It was found that the comet assay data did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiation types, 220 kV X rays and the gamma rays of 137Cs and 60Co, either for the total dose range or in the low-dose range. These results are, with some restrictions, consistent with physical examinations and predictions concerning, for example, the assessment of the possible difference in effectiveness in causing strand breaks between mammography X rays and conventional (150-250 kV) X rays, indicating that differences in biological effects must arise through downstream processing of the damage.     

Life shortening and disease incidence in BALB/c mice following a single d(50)-Be neutron or gamma exposure

Male BALB/c mice, 12 weeks old, were given a single exposure of either 137Cs gamma rays or d(50)-Be neutrons at a dose rate of 3 Gy/min. The animals were kept until death, and causes of death or possible causes of death were ascertained by autopsy and histology. The data were evaluated by competing risk methods. The survival time dose-effect curve for both types of exposure was linear and did not differ significantly (slopes: 55.8 +/- 4.0 days/Gy for neutrons and 46.2 +/- 4.3 days/Gy for gamma rays). The incidence of different diseases also was similar for both groups except that more carcinomas, sarcomas, and myeloid leukemias seemed to occur after neutron exposure and that nonstochastic lung and kidney diseases seemed to arise at lower doses.     

Cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with accelerated 56Fe ions

We examined cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with high-energy 56Fe ions. Cells were irradiated with graded doses of 56Fe ions (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The survival curves for cells plated 1 h after irradiation (immediate plating) showed little or no shoulder. However, the survival curves for cells plated 24 h after irradiation (delayed plating) had a small initial shoulder. The RBE for 56Fe ions compared to 137Cs gamma rays was 1.99 for immediate plating and 2.73 for delayed plating at the D10. The repair ratio (delayed plating/immediate plating) was 1.67 for 137Cs gamma rays and 1.22 for 56Fe ions. The dose-response curves for initially measured and residual chromatid fragments detected by the Calyculin A-mediated premature chromosome condensation technique showed a linear response. The results indicated that the induction frequency for initially measured fragments was the same for 137Cs gamma rays and 56Fe ions. On the other hand, approximately 85% of the fragments induced by 137Cs gamma rays had rejoined after 24 h of postirradiation incubation; the corresponding amount for 56Fe ions was 37%. Furthermore, the frequency of chromatid exchanges induced by gamma rays measured 24 h after irradiation was higher than that induced by 56Fe ions. No difference in the amount of chromatid damage induced by the two types of radiations was detected when assayed 1 h after irradiation. The results suggest that high-energy 56Fe ions induce a higher frequency of complex, unrepairable damage at both the cellular and chromosomal levels than 137Cs gamma rays in the target cells for radiation-induced lung cancers.     

Cytogenetic damage in lymphocytes for the purpose of dose reconstruction: a review of three recent radiation accidents

The analysis of chromosomal aberrations in peripheral blood of radiation accident victims is an established method of biological dosimetry. The dose estimate on the basis of an in vitro calibration curve is straightforward when the radiation exposure is homogeneous and the analysis not delayed. In recent years three radiation accidents occurred, where the irradiation or sampling conditions precluded a simple estimation of the dose. During the Georgian accident soldiers carried in their pockets small sources of 137Cs leading to partial and protracted body exposures. During the Tokai-mura accident, three employees involved in the process of 235U enrichment were exposed to very high doses of gamma rays and neutrons. During the Bialystok accident, five patients with breast cancer undergoing radiotherapy were exposed to a single dose of electrons which reached about 100 Gy. In the present paper the approaches chosen to estimate, by cytogenetic methods, the doses absorbed by the people involved in the accidents are described.     

The effects of a fractionated gamma irradiation on life shortening and disease incidence in BALB/c mice

BALB/c male mice (12 weeks old) were exposed to a single or fractionated exposure of 137Cs gamma rays. The fractionated dose was split into 10 equal doses delivered at an interval of 1 day. The causes and possible causes of spontaneous death were ascertained by autopsy and histological examination, and the data were treated by competing risk analysis. Life shortening followed a linear dose dependency and was about the same for fractionated (38.1 +/- 3.1 days/Gy) as for single (46.2 +/- 4.3 days/Gy) exposure. Death from tumor disease was enhanced and that from nonstochastic lung and kidney diseases was reduced after fractionated compared to single exposure.     

Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity.

The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.     

Radioprotective effects of dimethyl sulfoxide in golden hamster embryo cells exposed to gamma rays at 77 K. II. Protection from lethal, chromosomal, and DNA damage

Golden hamster embryo cells were exposed to 137Cs gamma rays in the presence or absence of dimethyl sulfoxide at both 310 and 77 K. Dimethyl sulfoxide gave significant protection against cell killing at both 310 and 77 K. The extent of radioprotection with 1.28 M dimethyl sulfoxide at 77 K was 85-89% of the lethal effects observed in the absence of dimethyl sulfoxide at 310 K; the dose-modifying factor was 5.7. Dimethyl sulfoxide also exerted protected against gamma-ray-induced DNA single-strand breaks and chromosomal aberrations with a maximum protection of 80-100% at a dimethyl sulfoxide concentration of 1.28 M at 77 K. At 77 K, H atoms, ion holes, and electrons can migrate through frozen cells but OH radicals cannot diffuse. Thus the protective effects of dimethyl sulfoxide against cell killing, chromosomal aberrations, and DNA single-strand breaks at 77 K may be due to the scavenging of H atoms or other ions, rather than OH radicals.     

Biological effectiveness of accelerated particles for the induction of chromosome damage measured in metaphase and interphase human lymphocytes

Chromosome aberrations were investigated in human lymphocytes after in vitro exposure to 1H-, 3He-, 12C-, 40Ar-, 28Si-, 56Fe-, or 197Au-ion beams, with LET ranging from approximately 0.4-1393 keV/microm in the dose range of 0.075-3 Gy. Dose-response curves for chromosome exchanges, measured at the first mitosis postirradiation using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosomal damage with respect to low- or high-dose-rate gamma rays. Estimates of RBEmax values for mitotic spreads, which ranged from near 0.7 to 11.1 for total exchanges, increased with LET, reaching a maximum at about 150 keV/microm, and decreased with further increase in LET. RBEs for complex aberrations are undefined due to the lack of an initial slope for gamma rays. Additionally, the effect of mitotic delay on RBE values was investigated by measuring chromosome aberrations in interphase after chemically induced premature chromosome condensation (PCC), and values were up to threefold higher than for metaphase analysis.     

Paternally inherited effects of gamma radiation on mouse preimplantation development detected by the chimera assay

It has previously been shown that type B spermatogonia in male mice treated with 0.05 Gy of X rays undergo an alteration expressed by progeny embryos as a cellular proliferation disadvantage in a chimera assay. We wished to obtain information on the assay's detection limit to ionizing radiation and on the radiosensitive target in male germ cells. Male mice were briefly irradiated with 137Cs gamma rays at nominal absorbed doses of 0.0, 0.0015, 0.005, 0.010, or 0.05 Gy and then mated for the next 8 weeks to untreated females. Four-cell embryos from treated males (experimental embryos) were paired with FITC-labeled embryos from untreated males (control embryos) to form aggregation chimeras. The chimeras were cultured for 30-40 h and examined under phase-contrast and UV illumination for the number of unlabeled cells (from the experimental embryo) and total chimera cell number, which were then expressed as "proliferation ratios" (No. unlabeled cells/total chimera cell No.). Significant decreases in proliferation ratios were observed at postirradiation weeks 4, 6, and 7 for the 0.01-Gy dose group and at weeks 5-6 for the 0.05-Gy dose group. In addition, significantly lower ratios were observed with early and mid four-cell embryos, but not with late four-cell embryos. These results suggest that mouse male germ cells express a radiosensitive target(s) whose detection limit by the assay lies at an absorbed dose between 0.005 and 0.010 Gy for brief gamma irradiation and whose effect on embryonic cell proliferation might decay by the second cleavage.     

Dose-dependent changes in the spectrum of mutations induced by ionizing radiation

We examined the influence of dose on the spectrum of mutations induced at the hypoxanthine guanine phosphoribosyltransferase (Hprt) locus in Chinese hamster ovary (CHO) cells. Independent CHO-K1 cell mutants at the Hprt locus were isolated from cells exposed to 0, 0.5, 1.5, 3.0 and 6.0 Gy (137)Cs gamma rays, and the genetic changes responsible for the mutations were determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. We observed dose-dependent changes in mutation spectra. At low doses, the principal radiation-induced mutations were point mutations. With increasing dose, multibase deletion mutations became the predominant mutation type such that by 6.0 Gy, there were almost three times more deletion mutations than point mutations. The dose response for induction of point mutations was linear while that for multibase deletions fit a linear-quadratic response. There was a biphasic distribution of deletion sizes, and different dose responses for small compared to large deletions. The frequency of large (>36 kb) total gene deletions increased exponentially, implying that they develop from the interaction between two independent events. In contrast, the dose response for deletion mutations of less than 10 kb was nearly linear, suggesting that these types of mutations develop mostly from single events and not the interactions between two independently produced lesions. The observation of dose-dependent changes in radiation-induced mutation spectra suggests that the types of alterations and therefore the risks from low-dose radiation exposure cannot be easily extrapolated from high-dose effects.     

Suppression of thymic lymphoma induction by life-long low-dose-rate irradiation accompanied by immune activation in C57BL/6 mice

The induction of thymic lymphomas by whole-body X irradiation with four doses of 1.8 Gy (total dose: 7.2 Gy) in C57BL/6 mice was suppressed from a high frequency (90%) to 63% by preirradiation with 0.075 Gy X rays given 6 h before each 1.8-Gy irradiation. This level was further suppressed to 43% by continuous whole-body irradiation with 137Cs gamma rays at a low dose rate of 1.2 mGy/h for 450 days, starting 35 days before the challenging irradiation. Continuous irradiation at 1.2 mGy/h resulting in a total dose of 7.2 Gy over 258 days yielded no thymic lymphomas, indicating that this low-dose-rate radiation does not induce these tumors. Further continuous irradiation up to 450 days (total dose: 12.6 Gy) produced no tumors. Continuously irradiated mice showed no loss of hair and a greater body weight than unirradiated controls. Immune activities of the mice, as measured by the numbers of CD4+ T cells, CD40+ B cells, and antibody-producing cells in the spleen after immunization with sheep red blood cells, were significantly increased by continuous 1.2-mGy/h irradiation alone. These results indicate the presence of an adaptive response in tumor induction, the involvement of radiation-induced immune activation in tumor suppression, and a large dose and dose-rate effectiveness factor (DDREF) for tumor induction with extremely low-dose-rate radiation.     

Dose-rate effects of mammary tumor development in female Sprague-Dawley rats exposed to X and gamma radiation

Mammary tumour development was followed in two experiments involving a total of 2229 female Sprague-Dawley rats exposed to various doses of X or gamma rays at different dose rates. The data for another 462 rats exposed to tritiated water in one of these experiments were also analyzed. The incidence of adenocarcinomas and fibroadenomas at a given time after exposure increased linearly in proportion to total radiation dose for most groups. However, no significant increase in adenocarcinomas was observed with chronic gamma exposures up to 1.1 Gy, and the increase in fibroadenomas observed with chronic gamma exposures at a dose rate of 0.0076 Gy h-1 up to an accumulated dose of 3.3 Gy was small compared to that observed after acute exposures. The incidence of all mammary tumors increased almost linearly with the log of dose rate in the range 0.0076 to 26.3 Gy h-1 for 3 Gy total dose of gamma rays. The effects of X rays appeared to be less influenced by dose rate than were the effects of gamma rays.