The relative biological effectiveness of low doses of 14 MeV neutrons in steady-state murine spermatogenesis as determined by flow cytometry

The relative biological effectiveness of 14 MeV neutrons in the low-dose range < or =1 Gy has been determined in differentiating and differentiated spermatogonia. Male NMRI mice were exposed to single doses of 2 cGy to 3 Gy of (60)Co gamma rays or neutrons. The ratios of testicular S-phase cells, 4c primary spermatocytes, and elongated spermatids were quantified by DNA flow cytometry 2 to 70 days after irradiation and were found to decrease. Histological samples and testis weight were analyzed in parallel. Doses of 2-5 cGy neutrons and 10-50 cGy gamma rays significantly (P<0.05) decreased the proportions of S-phase cells, spermatocytes and elongated spermatids at 4, 14 and 28 days postirradiation. For S-phase cells, the biphasic shape of the cell survival curves was described with a D(50) of 5 cGy neutrons. The D(50) for (60)Co gamma rays and the relative biological effectiveness could not be determined. The relative biological effectiveness of neutrons at 50% reductions of testis weight, primary spermatocytes, and elongated spermatids were 2.5, 10.0 and 6.1, respectively. This in vivo assay is interesting because of its sensitivity at dose ranges that are relevant for exposures in the environment, the workplace and radiotherapy.     

Effects of single and split doses of cobalt-60 gamma rays and 14 MeV neutrons on mouse stem cell spermatogonia

The long-term effects of ionizing radiation on male gonads may be the result of damage to spermatogonial stem cells. Doses of 10 cGy to 15 Gy (60)Co gamma rays or 10 cGy to 7 Gy 14 MeV neutrons were given to NMRI mice as single or split doses separated by a 24-h interval. The ratios of haploid spermatids/2c cells and the coefficients of variation of DNA histogram peaks as measures of both the cytocidal and the clastogenic actions of radiation were analyzed by DNA flow cytometry after DAPI staining. The coefficient of variation is not only a statistical examination of the data but is also used here as a measure of residual damage to DNA (i.e. a biological dosimeter). Testicular histology was examined in parallel. At 70 days after irradiation, the relative biological effectiveness for neutrons at 50% survival of spermatogonial stem cells was 3.6 for single doses and 2.8 for split doses. The average coefficient of variation of unirradiated controls of elongated spermatids was doubled when stem cells were irradiated with single doses of approximately 14 Gy (60)Co gamma rays or 3 Gy neutrons and observed 70 days later. Split doses of (60)Co gamma rays were more effective than single doses, doubling DNA dispersion at 7 Gy. No fractionation effect was found with neutrons with coefficients of variation.     

The effect of graded doses of fission neutrons or X rays on the lymphoid compartment of the thymus in mice

Young adult CBA/H mice were exposed to graded doses of whole-body irradiation with either fast fission neutrons or 300 kVp X rays at center-line dose rates of 0.1 and 0.3 Gy/min, respectively. Dose-response curves were determined at Days 2 and 5 after irradiation for the total thymic cell survival and for the survival of thymocytes defined by monoclonal anti-Thy-1, -Lyt-1, -Lyt-2, and -T-200 antibodies as measured by flow cytofluorometric analysis. Cell dose-response curves of thymocytes show, 2 days after irradiation, a two-component curve with a radiosensitive part and a part refractory to irradiation. The radiosensitive part of the dose survival curve of the Lyt-2+ cells, i.e., mainly cortical cells, has a D0 value of about 0.26 and 0.60 Gy for neutrons and X rays, respectively, whereas that of the other cell types has corresponding D0 values of about 0.30 and 0.70 Gy. The radiorefractory part of the dose-response curves cannot be detected beyond 5 days after irradiation. At that time, the Lyt-2+ cells are again most radiosensitive with a D0 value of 0.37 and 0.99 Gy for neutrons and X rays, respectively. The other measured cell types have corresponding D0 values of about 0.47 Gy. The fission neutron RBE values for the reduction in the thymocyte populations defined by either monoclonal anti-Thy-1, -Lyt-1, -Lyt-2, or -T-200 antibodies to 1.0% vary from 2.6 to 2.8. Furthermore, the estimated D0 values of the Thy-1-, T-200- intrathymic precursor cells which repopulate the thymus during the bone marrow independent phase of the biphasic thymus regeneration after whole-body irradiation are 0.64-0.79 Gy for fission neutrons and 1.32-1.55 Gy for X rays.     

Effects of single gamma-irradiation on the activity of ornithine decarboxylase in the thymus and pulmonary tissue]

In studying the dose (0.1-6 Gy) and time (2 h to 180 days) dependence of ornithine decarboxylase activity, it was found that deviations from the control were more pronounced in the thymus than in the pulmonary tissue. The radiation effect was a function of dose and time after irradiation. A nonmonotonous type of the dose-response curve was observed 7 days after irradiation: the radiation effect with a low dose (0.1 Gy) was opposite to that with sublethal doses (1-6 Gy).     

Chromosome aberration frequencies produced by a 70-MeV proton beam

The effectiveness of a 70-MeV proton beam in the induction of chromosome aberrations was studied. We employed peripheral lymphocytes and analyzed the frequencies of dicentrics and rings after irradiation at doses ranging from 0.1 to 8.0 Gy at various depths within a Lucite phantom. The frequency of chromosome aberrations after irradiation with an unmodulated proton beam at 5 mm showed a dose-response relationship similar to that of 60Co gamma rays. However, irradiation at greater depths with the spread-out Bragg peak induced higher aberration frequencies at doses lower than those with gamma rays. Furthermore, the distribution curve of chromosome aberration frequencies as a function of depth was found to be slightly different from the physically measured depth-dose curve. With the spread-out Bragg peak the biological effects were more marked at greater depths, resulting in a distribution of relative biological effectiveness values. The results obtained from chromosome aberration analysis may not be related directly to those for the relationship between dose and cell killing. Slight differences in values for relative biological effectiveness due to the change of dose and site of proton beam irradiation may not be important for practical proton beam therapy, but may be important in the prevention of late radiation injuries.     

Immunochemical detection of DNA damage induction and repair at different cellular stages of spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation

An immunochemical method has been used to detect quantitatively DNA damage caused by ionizing radiation in germ cells. With this method, DNA strand breaks as well as lesions converted into breaks in alkaline medium are measured as a function of controlled partial unwinding of the DNA, a time-dependent process starting at each breakage site, followed by the determination of the relative amount of single-stranded regions by use of a single-strand specific monoclonal antibody. With this method the induction and repair of DNA damage in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids) of the hamster were investigated. Germ cells were irradiated in vitro with 60Co-gamma-rays, at doses between 0 and 5 Gy. A linear dose-response relationship was observed. Spermatocytes and round spermatids had normal, fast repair of the lesions when compared with the repair of these sites in cultured V79 or CHO cells and human lymphocytes. The elongated spermatids, however, showed hardly any repair. Similar results were obtained after the in vivo gamma-irradiation of hamsters with doses of 0. 4, and 8 Gy and subsequent isolation of germ cells. The damage was still detectable in the elongated spermatids at 24 h after exposure. The results of the experiments show substantial differences in repair capacity between different stages of germ cell development. Because DNA is the major target for mutation induction, this assay may be useful for assessment of the genetic risk of exposure of male germ cells to ionizing radiation, in relation to the stage of development.     

The re-establishment of hypersensitive cells in the crypts of irradiated mouse intestine

Within 3-6 h of small doses of radiation (gamma-rays) the number of dead cells (apoptotic cells) in the crypts of the small intestine reaches peak values. These return to normal levels only after times later than 1 day. After higher doses elevated levels of cell death persist for longer times. The dead cells first occur most frequently at the lower positions of the crypt (median value for the distribution of apoptotic fragments is about cell position 6). At later times more dead cells are observed at higher positions. Two doses of radiation separated by various time intervals have been used to investigate when after irradiation the cell population susceptible to acute cell death is re-established. Dead cells were scored 3 or 6 h after the second dose. The yield of dead cells after two doses represents the sum of the dead cells produced by, and persisting from, the first dose and new apoptotic cells induced by the second dose. Since the temporal and dose-dependence aspects of the dead-cell yield after the first dose alone is known, the additional dead cells attributable to the second dose alone can be determined by subtraction. Within 1-2 days of small doses (0.5 Gy) the sensitive cells, recognized histologically as apoptotic cells, are re-established at the base of the crypt (around cell position 6). After higher doses (9.0 Gy) they are not re-established until about the fourth day after irradiation. Even in the enlarged regenerating crypts the sensitive cells are found at the same position at the crypt base. It has been estimated that the crypt contains five or six cells that are susceptible to low doses (0.5 Gy) (hypersensitive cells) and up to a total of only seven or eight susceptible cells that can be induced by any dose to enter the sequence of changes implicit in apoptosis. Between 4 and 10 days after an initial irradiation of 9.0 Gy the total number of susceptible cells increased from seven to eight to about 10 to 13 per crypt.     

Effect of gamma-radiation on contents of lipid peroxidation products in animal blood

The dynamics of malonic dialdehyde (MD) dependence in the blood serum of Vistar line male rats on the intensity of irradiation after a total single irradiation with 60Co gamma-quantums in 1.0 and 5.0 Gy doses (during 30 days); 9.0 Gy (during 15 days) were studied. The MD contents values both in the norm and in 0.5, 1, 2, 4, 6, 8, 10, 15, 20 and 30 days after irradiation are given. The experimental data demonstrated the different sensitivity of the final LPO parameters to the doses of 1.0, 5.0, 9.0 Gy radiation depending on the dose power, the changes of these parameters being kept for a long time after irradiation.     

The effect of graded doses of fission neutrons or X rays on the stromal compartment of the thymus in mice

The effect of irradiation on the supportive role of the thymic stroma in T cell differentiation was investigated in a transplantation model using athymic nude mice and transplanted irradiated thymuses. In this model, neonatal CBA/H mice were exposed to graded doses of whole-body irradiation with fast fission neutrons of 1 MeV mean energy or 300 kVp X rays. The doses used varied from 2.75 up to 6.88 Gy fission neutrons and from 6.00 up to 15.00 Gy X rays at center-line dose rates of 0.10 and 0.30 Gy/min, respectively. Subsequently, the thymus was excised and a thymus lobe was transplanted under the kidney capsule of H-2 compatible nude mice. One and two months after transplantation, the T cell composition of the thymic transplant was investigated using immunohistology with monoclonal antibodies directed to the cell surface differentiation antigens Thy-1, Lyt-1, Lyt-2, MT-4, and T-200. Furthermore, the stromal cell composition of the thymic transplant was investigated with monoclonal antibodies directed to MHC antigens and with monoclonal antibodies defining different subsets of thymic stromal cells. To investigate the reconstitution capacity of the thymic transplant, the peripheral T cell number was measured using flow cytofluorometric analysis of nude spleen cells with the monoclonal antibodies anti-Thy-1, anti-Lyt-2, and anti-MT-4. The results of this investigation show that a neonatal thymus grafted in a nude mouse has a similar stromal and T cell composition as that of a normal thymus in situ. In addition, grafting of such a thymus results in a significant increase of the peripheral T cell number. Irradiation of the graft prior to transplantation has no effects on the stromal and T cell composition but the graft size decreases. This reduction of size shows a linear dose-response curve after neutron irradiation. The X-ray curve is linear for doses in excess of 6.00 Gy. The RBE for fission neutrons for the reduction of the relative thymic graft size to 10% was equal to 2.1. Furthermore, the peripheral T cell number decreases with increasing doses of irradiation given to the graft prior to transplantation. The present data indicate that the regenerative potential of thymic stromal cells is radiosensitive and is characterized by D0 values equal to 2.45 and 3.68 Gy for neutrons and X rays, respectively. In contrast, the ability of the thymic stromal cells to support T cell maturation is highly radioresistant.     

Low doses of X-rays decrease the risk of diploidy in mouse oocytes

Females from the NMRI/Han mouse strain ovulate a high number of diploid oocytes (about 12%) after gonadotrophin-stimulated ovulation. These oocytes can be fertilized and develop into triploid embryos subsequently. The exposure of such gonadotrophin-primed females to X-ray doses of 0.05, 0.10, 0.20 or 0.40 Gy during the preovulatory period (2 h after the HCG dose) significantly decreased the percentage of diploid oocytes. After the highest dose used, i.e. 0.80 Gy, however, the incidence was on the level from unirradiated females, again. We suggest that the observed negative hump-shaped dose response of diploidy is not caused by secondary modifications induced by irradiation, such as a selective killing of diploid oocytes before ovulation, or a (compensatory) super-ovulation of only normal oocytes, but rather is caused by a direct radiobiological interference of low doses in protecting from gonadotrophin-induced aneuploidy.     

Langerhans cell number and morphology in mouse footpad epidermis after X irradiation

Effects of 250-kV X rays on epidermal Langerhans cells were studied in CBA/CaH mice. One group received 20 Gy to the feet, another 8 Gy to the whole body, and a third both the 8 Gy whole-body and a 12 Gy local dose to the feet. Mice from each group and controls were sacrificed at intervals from 1 to 64 days later. ATPase-positive cells in sheets of footpad epidermis were counted by light microscopy. The density of Langerhans cells in controls was 1515 +/- 36/mm2 (mean +/- SE; n = 34). By 3 days after irradiation they became rounded and less dendritic and numbers gradually reached a nadir by 10 days, at 18% of controls after 20 Gy and 57% of controls after 8 Gy. Some of the remainder exhibited bizarre morphology and ultrastructural abnormalities. After local irradiation of the feet Langerhans cell numbers recovered rapidly between 14 and 16 days, although their distribution was uneven until 30 days after irradiation. Repopulation was delayed after an 8 Gy whole-body dose by at least 3 weeks. These results demonstrate that high local doses of X rays substantially but transiently deplete the epidermal Langerhans cell population and support the hypothesis that functional hemopoietic tissue is required for extensive Langerhans cell replenishment.     

Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions.

Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.     

Immunochemical detection of DNA damage induction and repair at different cellular stages of spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation

An immunochemical method has been used to detect quantitatively DNA damage caused by ionizing radiation in germ cells. With this method, DNA strand breaks as well as lesions converted into breaks in alkaline medium are measured as a function of controlled partial unwinding of the DNA, a time-dependent process starting at each breakage site, followed by the determination of the relative amount of single-stranded regions by use of a single-strand specific monoclonal antibody. With this method the induction and repair of DNA damage in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids) of the hamster were investigated. Germ cells were irradiated in vitro with ⁶⁰Co-γ-rays, at doses between 0 and 5 Gy. A linear dose-response relationship was observed. Spermatocytes and round spermatids had normal, fast repair of the lesions when compared with the repair of these sites in cultured V79 or CHO cells and human lymphocytes. The elongated spermatids, however, showed hardly any repair. Similar results were obtained after the in vivo γ-irradiation of hamsters with doses of 0, 4, and 8 Gy and subsequent isolation of germ cells. The damage was still detectable in the elongated spermatids at 24 h after exposure. The results of the experiments show substantial differences in repair capacity between different stages of germ cell development. Because DNA is the major target for mutation induction, this assay may be useful for assessment of the genetic risk of exposure of male germ cells to ionizing radiation, in relation to the stage of development.     

Evaluation of the expression of damage of the DNA-structural complex of rat thymocytes based on kinetics of alkaline lysis following irradiation

Rotary viscosimeters were used to study the postirradiation destruction of the DNA-structural complex (DSC) of rat thymocyte nuclei exhibited by a change in alkaline denaturation of DSC upon lysis. The S area, limited by the characteristic viscosity values obtained during alkaline lysis of thymocyte nuclei, was used as a characteristic of DSC. Immediately after irradiation the S area changed up to 81-84 per cent at 0.5-1.5 Gy and up to 56-44 per cent at 2-10 Gy. 6 to 24 h following irradiation a change in the profile of alkaline denaturation of DSC was a function of dose and dropped from 100 down to 11 per cent at doses of 0 to 10 Gy. After 2-3 days, the changes in S were also observed but they were not a strict function of dose and were the same with the values obtained immediately after irradiation.     

Effects of low-dose neutrons applied at reduced dose rate on human melanoma cells

Human melanoma cells that are resistant to gamma rays were irradiated with 14 MeV neutrons given at low doses ranging from 5 cGy to 1.12 Gy at a very low dose rate of 0.8 mGy min(-1) or a moderate dose rate of 40 mGy min(-1). The biological effects of neutrons were studied by two different methods: a cell survival assay after a 14-day incubation and an analysis of chromosomal aberrations in metaphases collected 20 h after irradiation. Unusual features of the survival curve at very low dose rate were a marked increase in cell killing at 5 cGy followed by a plateau for survival from 10 to 32.5 cGy. The levels of induced chromosomal aberrations showed a similar increase for both dose rates at 7.5 cGy and the existence of a plateau at the very low dose rate from 15 to 30 cGy. The existence of a plateau suggests that a repair process after low-dose neutrons might be induced after a threshold dose of 5-7.5 cGy which compensates for induced damage from doses as high as 32.5 cGy. These findings may be of interest for understanding the relative biological effectiveness of neutrons and the effects of environmental low-dose irradiation.     

A comparison of gamma and neutron irradiation on Raji cells: effects on DNA damage, repair, cell cycle distribution and lethality.

The Comet assay (microgel electrophoresis) was used to study DNA damage in Raji cells, a B-lymphoblastoid cell line, after treatment with different doses of neutrons (0.5 to 16 Gy) or gamma rays (1.4 to 44.8 Gy). A better growth recovery was observed in cells after gamma-ray treatments compared with neutron treatments. The relative biological effectiveness (RBE) of neutron in cell killing was determined to be 2.5. Initially, the number of damaged cells per unit dose was approximately the same after neutron and gamma-ray irradiation. One hour after treatment, however, the number of normal cells per unit dose was much lower for neutrons than for gamma rays, suggesting a more efficient initial repair for gamma rays. Twenty-four hours after treatment, the numbers of damaged cells per unit dose of neutrons or gamma rays were again at comparable level. Cell cycle kinetic studies showed a strong G2/M arrest at equivalent unit dose (neutrons up to 8 Gy; gamma rays up to 5.6 Gy), suggesting a period in cell cycle for DNA repair. However, only cells treated with low doses (up to 2 Gy) seemed to be capable of returning into normal cell cycle within 4 days. For the highest dose of neutrons, decline in the number of normal cells seen at already 3 days after treatment was deeper compared with equivalent unit doses of gamma rays. Our present results support different mechanisms of action by these two irradiations and suggest the generation of locally multiply damaged sites (LMDS) for high linear energy transfer (LET) radiation which are known to be repaired at lower efficiency.     

Effect of ionizing radiation on Fc-receptors of mouse peritoneal mononuclear cells

The influence of different gamma-radiation doses on the number of mouse peritoneal mononuclears bearing Fc-receptors was studied. At doses of 7 and 9.5 Gy, phase changes were observed in the number of cells bearing Fc-receptors: a short-term rise registered 24 h to 2 days after irradiation was followed by depression. In addition, the total number of peritoneal mononuclears decreased. At a dose of 100 Gy, the number of cells with Fc-receptors was reduced as early as 1 h after irradiation, and the pronounced cell lysis occurred.     

Genetic consequences of irradiation of one or both parents (results of experiments on Wistar rats) Exitus lethalis in Wistar rat's progeny after irradiation of one or both parents

Examination of 2563 offsprings of Wistar rats after irradiation of one or both parents with doses of 0.25, 0.5, 1, 2, 3 and 4 Gy was carried out; the manifestation of lethal effects in the progeny of the first generation in ontogenesis was studied. The level of embryonic death was the highest after irradiation of germ cells of parents at stages of spermatids, spermatozoids and matured oocytes. Following irradiation of both parents with doses of 0.25, 0.5 and 1 Gy at these stages of gametogenesis and 4 Gy at the stage of spermatids and matured oocytes there was a trend of increasing radiation effects caused by the participation of two irradiated germ cells. After irradiation of both parents with doses of 2, 3 and 4 Gy the embryonic death F1 was essentially the same as rates for irradiated females and non-irradiated males. The F1 death rate in early postnatal development exceeded the control only after irradiation with doses of 2, 3 and 4 Gy. The increase in radiation effects in the F1 due to the mating of two irradiated parents appears to be associated with a mechanism demonstrating additivity or synergism.     

Threshold-like dose of local beta irradiation repeated throughout the life span of mice for induction of skin and bone tumors

The backs of female ICR mice were irradiated with beta rays from 90Sr-90Y three times a week throughout life. Previously we observed 100% tumor incidence at five different dose levels ranging from 1.5 to 11.8 Gy per exposure, but no tumor on repeated irradiation with 1.35 Gy for 300 days (Radiat. Res. 115, 488, 1988). In the present study, delay of tumor development was again seen at a dose of 1.5 Gy per exposure, with further delay at 1.0 Gy. The final tumor incidence was 100% with these two doses. At 0.75 Gy per exposure, no tumor appeared within 790 days after the start of irradiation, but one osteosarcoma and one squamous cell carcinoma did finally appear. These findings indicate a threshold-like response of tumor induction in this repeated irradiation system and further suggest that the apparent threshold may be somewhat less than 0.75 Gy per exposure.     

Effect of gamma-radiation on the content of vitamin E in rats

The dynamics of vitamin E dose-dependence in the blood serum of Vistar line male rats after the total single irradiation with 60Co gamma-quantums in 0.2; 0.5; 1.0; 3.0 and 5.0 Gy doses (during 120 days); 7.0 Gy (during 20 days); 9.0 Gy (during 15 days) were studied. The vitamin E content values both in the norm and in 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 30, 45, 65, 90 and 120 days after irradiation are given. The experiment data show that the dose dependence of vitamin E products has wavelike character. The possible causes of the vitamin E level decrease under intake of small and high doses of gamma-irradiation are discussed.