Vascular and epithelial damage in the lung of the mouse after X rays or neutrons

The response of the lung was studied in CFLP mice after exposure of the whole thorax to X rays (250 kVp) or cyclotron neutrons (16 MeV deuterons on Be, mean energy 7.5 MeV). To measure blood volume and leakage of plasma proteins, 51Cr-labeled red blood cells and 125I-albumin were injected intravenously and 24 h later lungs were lavaged via the trachea. Radioactivities in lung tissue and lavage fluid were determined to estimate the accumulation of albumin in the interstitial and alveolar spaces indicating damage to blood vessels and alveolar epithelium respectively. Function of type II pneumonocytes was assessed by the amounts of surfactant (assayed as lipid phosphorous) released into the lavage fluid. During the first 6 weeks, lavage protein and surfactant were increased, the neutron relative biological effectiveness (RBE) being unity. During pneumonitis at 12-24 weeks, surfactant levels were normal, blood volume was decreased, and both interstitial and alveolar albumin were increased. Albumin levels then decreased. At late times after exposure (42-64 weeks) alveolar albumin returned to normal but interstitial albumin was still slightly elevated. Values of RBE for changes in blood volume and interstitial and alveolar albumin at 15 weeks and for changes in blood volume and interstitial albumin at 46 weeks were 1.4, comparable with that for animal survival at 180 days. The results indicate that surfactant production is not critical for animal survival. They suggest that changes in blood vessels and alveolar epithelium occur during acute pneumonitis; epithelial repair follows but some vascular damage may persist. The time course of the changes in albumin levels did not correlate with increases in collagen biosynthesis which have been observed as early as 1 month after exposure and persist for up to 1 year. Furthermore, a dose which had no effect on leakage caused a marked increase in collagen biosynthesis. Thus the present results do not support a causal relationship between exudation of vascular protein during pneumonitis and the later development of fibrosis.     

Induction of preneoplastic alterations by X rays and neutrons in exposed rat tracheas and isolated tracheal epithelial cells

The relative potential of high- and low-LET radiation to induce preneoplastic alterations in rat tracheal epithelial cells was evaluated using a combined in vivo-cell culture model. The capacity of X rays and high- and low-dose-rate neutrons to induce preneoplastic changes in isolated rat tracheal epithelial cells and in the intact tissue was compared. The presence of altered populations was determined in culture in terms of the frequency of tracheal epithelial cell populations which exhibit enhanced growth capacity in culture and in terms of the induction of persistent morphological alterations in exposed transplanted tracheas. Prior to assaying for altered cells, tracheal epithelial cells were irradiated as part of the intact tissue or as single cells. Low- and high-LET radiation induced similar maximum frequencies of altered cells when cultures were exposed as single cells, although high-LET radiation was more radiobiologically effective (RBE = 20) than low-LET radiation. The most marked difference between high- and low-LET radiation was observed after irradiation of the intact tissue. Damage induced by low-LET radiation, giving rise to altered populations, was modified in the intact tissue, whereas similar damage induced by high-LET radiation was apparently not.     

Clonogenic cells and rat mammary cancer: effects of hormones, X rays, and fission neutrons

On Day 0, young adult female F344 rats were adrenalectomized and intrasplenically implanted with a pituitary gland and capsule containing estrone. All were thereafter given 2.5 mg deoxycorticosterone per week and the choice of saline or tap water. This treatment yields high prolactin levels and glucocorticoid deficiency (Prl+/Glc-). On Day +48, total recoverable mammary DNA was increased by more than sevenfold, tritiated thymidine uptake by nearly fourfold, and total mammary clonogens by about fivefold. Irradiation with 4, 40, and 80 cGy X rays on Day +48 increased total mammary carcinomas per rat day at risk linearly with dose, and 40 and 80 cGy significantly decreased first carcinoma latency. A dose of 40 cGy X rays on Day -1 yielded tumor latencies and frequencies insignificantly different from unirradiated controls and significantly different from the dose on Day +48. Total carcinomas per rat day at risk were better fit by a function of dose to the power 0.4 than by a linear function after exposure to 1, 10, and 20 cGy fission neutrons, and 10 and 20 cGy significantly shortened the time to appearance of the first cancer. In contrast to results with X rays, 10 cGy neutrons on Day -1 yielded tumor frequencies and latencies insignificantly different from 10 cGy neutrons on Day +48. The carcinogenic action of X rays was thus influenced by total clonogen numbers and/or proliferation rates; that of neutrons was not.     

Short- and long-term effects of whole-body irradiation with fission neutrons or X rays on the thymus in CBA mice

Young adult (6 weeks old) female CBA mice were exposed to whole-body irradiation with either 2.5-Gy fast fission neutrons of 1 MeV mean energy or 6.0-Gy 300 kVp X rays at centerline dose rates of 0.1 and 0.3 Gy/min, respectively. The weight of spleen and animal and the weight, cellularity, and histological structure of the thymus were studied at different times after irradiation. Thymic recovery after whole-body irradiation showed a biphasic pattern with minima at 5 and 21 days after irradiation and peaks of regeneration at Days 14 and 42 after X irradiation or at Days 14 and 70 after neutron irradiation. After the second phase of recovery, a marked decrease in relative thymus weight and cellularity was observed, which lasted up to at least 250 days after irradiation. Splenic recovery showed a monophasic pattern with an overshoot on Day 21 after irradiation. After neutron irradiation a late decrease in relative spleen and animal weight was observed. The observed late effects on thymus and spleen weight and thymus cellularity are discussed in terms of a persistent defect in the bone marrow.     

Characteristics of chromatin breakdown in the rat thymus after exposure to fast neutrons and gamma rays

A comparative study was made of the radiobiological aftereffects of the action of fast neutrons and gamma-rays on lymphoid tissues of rat thymus with a reference to a biochemical criterion of the interphase death of lymphocytes, i.e. the formation of polydeoxynucleotides (PDN). It was shown that the increase in the chromatin degradation was a function of dose of neutron- and gamma-radiation (up to 4 Gy). The dynamics of the PDN formation was similar with both types of radiation, but 4-6 h after neutron irradiation chromatin degradation was higher more pronounced. The RBE of neutrons varied from 3 to 2 with a radiation dose varying from 0.25 to 4 Gy.     

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.     

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.     

Microdosimetry and chromosome aberrations: Effects of 230 keV neutrons on Vicia faba chromosomes

Physical energy deposition events have been related to sub-nuclear cytological events (chromosomal changes) in metaphases sequentially accumulated from the latter part of the cell cycle of Vicia faba. 230 keV neutrons produce about 0.4 recoil protons per late interphase nucleus per rad with the majority of protons travelling 1 to 2 microns from their origin, depositing energy at around 90 keV per micron. The frequency of induced aberrations is basically linear with dose, though varying through consecutive cell sampling periods because of differential induced mitotic delay. Distributions of chromosomal aberrations and total cytological events are overdispersed in relation to the Poisson distribuyion indicating that some proton recoils produce multiple events. When gaps and aberrations within chromosomes and multiple aberrations between chromosomes, are considered as discrete events, distributions follow Poisson expectations. About 40% of proton recoils result in observable cytological change. The highly energetic proton recoils (～90 keV per micron) which can induce multiple events are the ones most likely to produce effects which result in cell death. The sphere of influence of the proton recoils is probably adequately estimated from their range (～1 to 2 μm) since it seems compatible with the spatial proximity of the initial components of the resultant chromosome aberrations.     

Induction of incomplete and complex chromosome aberrations by 30 kVp X rays

X rays of 26-30 kVp are routinely used for mammography screening. For radioprotection purposes, a quality factor (Q) of 1 is assumed for all photon energies, but it is thought that the relative biological effectiveness (RBE) increases as the photon energy decreases. The analysis of radiation-induced chromosome aberrations is one of the most widely used methods to study the interaction between radiation and DNA. Here we present a FISH study on metaphases from peripheral blood samples irradiated with three different X-ray energies (30, 80 and 120 kVp). The study comprises two FISH approaches: one using pantelomeric and pancentromeric probes to evaluate the induction of incomplete chromosome aberrations and the other using mFISH to evaluate the induction of complex chromosome aberrations. The results indicate that exposure to 30 kVp X rays resulted in a modest increase in the induction of incomplete elements and complex aberrations compared to 80 and 120 kVp X rays.     

The effects of graded doses of 1 MeV fission neutrons or X rays on the murine hematopoietic stroma.

The acute radiosensitivity in vivo of the murine hematopoietic stroma for 1 MeV fission neutrons or 300 kVp X rays was determined. Two different assays were used: (1) an in vitro clonogenic assay for fibroblast precursor cells (CFU-F) and (2) subcutaneous grafting of femora or spleens. The number of stem cells (CFU-S) or precursor cells (CFU-C), which repopulated the subcutaneous implants, was used to measure the ability of the stroma to support hemopoiesis. The CFU-F were the most radiosensitive, and the survival curves after neutron and X irradiation were characterized by D0 values of 0.75 and 2.45 Gy, respectively. For regeneration of CFU-S and CFU-C in subcutaneously implanted femora, D0 values of 0.92 and 0.84 Gy after neutron irradiation and 2.78 and 2.61 Gy after X irradiation were found. The regeneration of CFU-S and CFU-C in subcutaneously implanted spleens was highly radioresistant as evidenced by D0 values of 2.29 and 1.49 Gy for survival curves obtained after neutron irradiation, and D0 values of 6.34 and 4.85 Gy after X irradiation. The fission-neutron RBE for all the cell populations was close to 3 and varied from 2.77 to 3.28. The higher RBE values observed for stromal cells, compared to the RBE of 2.1 reported previously for hemopoietic stem cells, indicate that stromal cells are relatively more sensitive than hemopoietic cells to neutron irradiation.     

Conformational properties of DNA after exposure to gamma rays and neutrons

DNA aqueous solutions were irradiated with 0-40 Gy of 60Co gamma rays and 0-1.5 Gy of (Pu-Be) neutrons. Thermal transition spectrophotometry (TTS) was used to trace the changes in the DNA conformation at the above doses. Previous results using the perturbed angular correlation (PAC) method were used to complement to the current analysis. The TTS and PAC methods are two different approaches to the study of the effects of radiation on DNA. Both showed that neutrons are more effective than gamma rays in inducing DNA damage. The TTS method showed that neutrons are 11 +/- 5 times more efficient than gamma rays, while the PAC method had shown this value to be 34 +/- 4. From the current study we deduced that the radiation damage to DNA is not a spontaneous effect but rather is an ensemble of damaging events that occur asynchronously. Any single method selected for the study of such damages can concentrate on only a part of the damage, leading to over- or underestimation of the relative effectiveness of the neutrons.     

Analysis of loss of inactive X chromosomes in interphase cells.

We have developed a method that allows, for the first time, a specific analysis of the inactive X chromosome (Xi) in interphase cells. By combining immunolabeling of acetylated histone H4 with specific antisera and FISH with an X-chromosome centromere-specific DNA probe, micronucleated whole Xis in human female cells may be identified by their lack of histone H4 acetylation. As one example of the potential applications of this methodology in genetic studies in humans, an artifact-free X-chromosome aneuploidy detection in lymphocytes of women of different ages has been performed. Our results indicate that not only the Xi but also the active X chromosome is preferentially lost during aging, indicating that the high frequency of sex-chromosome aneuploidy in human females cannot be explained solely by a lack of negative selection of Xi aneuploid cells. Further applications of the proposed methodology in genetic studies are discussed.     

Segregation after mitotic crossing-over in isodicentric X chromosomes

Segregation after mitotic crossing-over in an isodicentric (idic) X chromosome with one active and one inactive centromere has given rise to two new cell lines, one in which the idic(Xpter) chromosome has two active centromeres (most of these chromosomes also have an inversion) and another in which neither centromere is active. The two X chromosomes are attached at the telomeres of their short arms. Similar segregation has given rise to two other cell lines with idic(Xq-) chromosomes. Other observations on segregation after mitotic crossing-over are reviewed. Unequal crossing-over has apparently played a major role in the evolution of various genes and heterochromatin. Retinoblastoma and Wilms tumor are in some cases associated with homozygosity of a chromosome segment resulting from mitotic crossing-over. Similarly, the high incidence of cancer in Bloom syndrome may be caused by mitotic crossing-over leading to homozygosity or amplification of oncogenes.     

Lung carcinomas in Sprague-Dawley rats after exposure to low doses of radon daughters, fission neutrons, or gamma rays

The effectiveness of radon-daughter inhalation and irradiation with fission neutrons and gamma rays in the induction of lung carcinomas in Sprague-Dawley rats at low doses is compared. Earlier reports which compared radon-daughter inhalations and neutron irradiations over a wider range of doses were based on dosimetry for the radon-daughter inhalations which has recently been found to be faulty. In the present analysis, low-dose experiments were designed to derive revised equivalence ratios between radon-daughter exposures, and fission neutron or gamma irradiations. The equivalence is approximately 15 working level months (WLM) of radon daughters to 10 mGy of neutrons (the earlier value was 30 WLM to 10 mGy). The relative biological effectiveness (RBE) of neutrons is 50 or more at a gamma-ray dose of 1 Gy. In these experiments with low doses and exposures, the lifetime incidences can be estimated from the raw incidences, while the derivation of the time dependence of the prevalence is essential for the estimation of RBE values and equivalence ratios.