The Intrinsic Radiosensitivity of Lymphocytes in Chronic Lymphocytic Leukaemia,Quantitatively Determined Independently of Cell Death Rate Factors

Summary

Survival curve shape for lymphocytes X-irradiated in vitro is governed by death rate as well as intrinsic radiosensitivity. We have resolved into these two components the survival curves obtained for CLL lymphocytes by use of a simple mathematical model. A multiple correlation coefficient comparing the predicted with the experimental survival curves was close to unity (0·954–0·999). For 14/18 patients with unequivocal B-cell CLL, the leukaemic (colchicine ultrasensitive) cells behaved as a homogenous population (D₃₇ 0·32–1·28 Gy). This is similar to the more radiosensitive class of lymphocytes of normal blood (believed to include the B cells) and is some 4-fold less than the more radioresistant class (comprising most of the T cells). The lethally hit cells were homogeneous in death rate, which followed first order kinetics. The half-life (range 9–87 h) was, on average, some 50 per cent shorter than the more radiosensitive normal lymphocytes. The remaining four patients constituted a miscellaneous group. From one of these, it can be seen that an excessively slow death rate can give the misleading impression of radioresistance. It is hypothesized that the benefit afforded certain CLL patients treated with low-dose total body irradiation (TBI) or splenic irradiation (SI) may reside, partly, in the sparing of T lymphocytes of the helper type and in accompanying selective elimination (or functional inactivation) of those of the suppressor type.     

Pseudouridine in Animal and Human Urine after Irradiation

Summary

The radioresistant tissues, heart, liver, lung and kidney, contain 2 to 15 times more protein per cell, nucleus and chromatin compared with the radiosensitive tissues, spleen and thymus. As determined by polyacrylamide gel electrophoresis, in the nuclear fraction and chromatin of radiosensitive tissues there is a deficit of high molecular weight (M_w > 20 000) non-histone proteins. It is suggested that the radiosensitivity of lymphoid cells, which die of interphase death a few hours after exposure to small doses of radiation, is closely correlated with this deficit in non-histone proteins.     

Lethal and Mutagenic Effects of 252Cf Radiation in Cultured Human Cells

Summary

HeLa MR cells were exposed to radiation emitted from a man-made spontaneously fissioning isotope, californium-252. The neutron to gamma-ray ratio in the radiation dose was measured to be 2·0. The extrapolation number of the dose-survival curve was 1·3 and the Do was 200 cGy. A dose-dependent increase in mutation to 6-TG^r (6-thioguanine resistant) was observed. The relative biological effectiveness (r.b.e.) for cell killing of the neutrons from ²⁵²Cf, calculated relative to high-dose-rate X-rays, was 2·6 at 50 per cent survival. The r.b.e. for mutation induction was 2·7 at a mutation frequency of 5 × 10^?5 per surviving cell.     

Chromosomal Abnormalities Induced by Iodine-125 in Mouse Germ Cells

Summary

Swiss albino male mice were injected intraperitoneally with 0, 185, 370 or 555 kBq (0, 5, 10 or 15 µCi) of iodine-125 (¹²⁵I). All the animals were killed on the sixtieth day and chromosomal aberrations were screened in spermatocytes at meiotic metaphase I. A significant increase in the percentage of chromosomal aberrations including translocations (0, 1·2, 1·8 and 2·3 per cent translocations in controls, 185, 370 and 555 kBq groups respectively) was recorded at all dose levels indicating the clatogenic effects of ¹²⁵I in mouse spermatocytes.     

Overview of Radiation-induced Skin Cancer in Humans

Summary

There are about a dozen studies of the incidence of skin cancer among irradiated populations with known skin doses that are available for estimating the risk of radiation-induced skin cancer. It is of note that they provide no evidence for a dose threshold and are compatible with a linear dose–response relationship, at least for ultraviolet radiation exposed skin. The studies also provide varying amounts of evidence concerning a number of other important issues in assessing skin cancer risk: types of skin cancer induced by ionizing radiation, the appropriateness of relative risk vs absolute risk models, combined effects of ionizing and UV radiations, and variations in sensitivity to skin cancer induction among demographic and genetic subgroups. Little epidemiological information is available on several factors, such as the RBE for high-LET radiation, the effects of dose protraction or fractionation, or variations in risk by age at irradiation. A reasonable estimate of skin cancer lethality was 0·2 per cent when weighted for the relative proportions of squamous cell and basal cell skin cancers. Average risk estimates of radiation-induced skin cancer incidence were: absolute risk (AR) of 8·5 × 10^?4 person-year-Sv and excess relative risk (RR) of 52 per cent/Sv. Lifetime skin cancer risk was calculated by life-table methods for males from exposures spread out over ages 20–60 years. The estimates for excess skin cancer incidence were 2 per cent and 11 per cent per Sv under the AR and RR models, respectively, while the corresponding mortality risks were 4 × 10^?5 and 2 × 10^?4 per Sv.     

The Effect of Dietary Phosphorus on the Metabolism of Calcium and Strontium in the Rat

Summary

An investigation has been made of the way in which changes in dietary phosphorus within the physiological range influence the comparative metabolism of calcium and strontium in the rat. Radioactive calcium and strontium were used as tracers.

The absorption of both calcium and strontium after oral administration was dependent on the phosphorus level in the diet, but not to the same degree. The skeletal ratio ⁸⁵Sr/⁴⁷Ca decreased by some 40 per cent as the dietary phosphorus was increased from 0·5 to 1·3 g per cent.

After intraperitoneal injection of radioactive calcium and strontium, the skeletal retention of ⁸⁵Sr was about 25 per cent more on a diet containing 1·3 per cent phosphorus than on a diet containing 0·5 per cent phosphorus. Skeletal retention of calcium varied little.

It is concluded that renal discrimination against strontium as well as intestinal absorption of calcium and strontium were affected by the phosphorus content of the diet, but in opposite directions.     

Abstracts of the Association for Radiation Research Winter Meeting,Leeds, 6–8 January 1988

Summary

A sensitive experimental design and data analysis were used to test rigorously whether the repair capacity in the skin of the mouse foot changes during a course of repeated 240 k Vp X-ray doses. Any such changes might reflect saturation or induction of repair enzymes resulting from progressive radiation damage, but most importantly this assumption of equal effect per dose fraction is central to all analyses of multiple-fraction radiation treatments, and remained to be demonstrated conclusively in skin. An X-ray dose of 2·5 Gy was given two, eight, 14 or 20 times with an interfraction interval of 8 h. Individual skin reactions for each mouse were analysed separately, giving 139 estimates of the effectiveness of 2·5 Gy (～ 35 in each of the four fractionation schedules). Regression analysis of effect per fraction versus number of fractions showed that there was no significant trend, i.e. the damage per fraction was constant regardless of the number of fractions used. The mean damage per fraction was 3·75 ± 0·15 per cent (95 per cent CL) of the full underlying damage equivalent to transient moist desquamation, and the slope of this plot was 0·0075 per cent ± 0·022 per cent (95 per cent CL) per fraction. It was concluded that the assumption of equal effect per fraction was not invalidated in mouse skin. Shorter interfraction intervals would not allow full repair between fractions, and this could be misinterpreted as a progressive loss of repair capacity in this type of experiment. This was tested in skin by giving 2·5 Gy doses two, eight and 14 times with a 1-h interfraction interval. Effect per fraction increased with number of fractions, by an extra 37 per cent from two to eight fractions and by a further 14 per cent from eight to 14 fractions, giving the illusion of loss of repair as predicted. This confirms the need to check that where loss of repair capacity is suspected, this is not due artifactually to incomplete repair between fractions in slowly repairing systems.     

Murine Haemopoietic Stem Cells with Long-term Engraftment and Marrow Repopulating Ability are More Resistant to Gamma-radiation than are Spleen Colony Forming Cells

The radiation sensitivity of various subsets in the haemopoietic stem cell hierarchy was defined using a limiting dilution type long-term bone marrow culture technique that was previously shown to allow quantification of cells with spleen colony-forming potential (day-12 CFU-S) and in vivo marrow repopulating ability (MRA). Primitive stem cells that generate new in vitro clonable colony-forming cells (CFU-C) in the irradiated marrow (MRA) and have long-term repopulation ability (LTRA) in vitro (cobblestone area forming cell, CAFC day-28) had D₀ values of 1·25 and 1·38 Gy, respectively. A lower D₀ was found for the less primitive CFU-S day-12, CAFC day-12 and cells with erythroid repopulating ability (0·91, 1·08 and 0·97 Gy, respectively). CFU-S day-7 were the most radiosensitive (D₀ equalling 0·79 Gy), while CFU-C and CAFC day-5 were relatively resistant to irradiation (D₀ 1·33 and 1·77 Gy). Split-dose irradiation with a 6 h interval gave dose sparing for stem cells with MRA and even more with in vitro LTRA, less for CFU-S day-12 and CAFC day-10 and none for CFU-S day-7. The cell survival data of the specified stem cell populations were compared with the ability of a fixed number of B6-Gpi-1^a donor bone marrow cells to provide for short- and long-term engraftment in single- and split-dose irradiated cognenic B6-Gpi-1^b mice. Serial blood glucose phosphate isomerase (Gpi) phenotyping showed less chimerism in the split as compared to the single radiation dose groups beyond 4 weeks after transplant. Radiation dose-response curves corresponding to stable chimerism at 12 weeks for single and fractionated doses revealed appreciable split-dose recovery (D₂–D₁) in the order of 2 Gy. This was comparable to D₂–D₁ estimates for MRA and late-developing CAFC (1·27 and 1·43 Gy, respectively), but differed from the poor dose recovery in cells corresponding to the committed CFU-S day-7/12 and CAFC day-10 population (0·14–0·33 Gy). These data are together consistent with differential radiosensitivity and repair in the haemopoietic stem cell hierarchy, and provide a cellular basis for explaining the dose-sparing effect of fractionated total-body irradiation conditioning on long-term host marrow repopulation.     

Measurement of the G-value for 1·5 keV X-rays

Summary

Although there are several theoretical predictions of the dependence of the G-value on X-ray energy, measurements have not been made below ≈ 7 keV. Using a ferrous sulfate solution modified by the addition of benzoic acid, we have measured the relative G-values for Al_k characteristic X-rays (1·5 keV), ²³⁸Pu α-particles (3·7 MeV), ⁶⁰Co (1·17 MeV) and ¹³⁷Cs (0·66 MeV) γ-rays. This modified ferrous sulfate solution gave a 4-fold increase in sensitivity relative to the conventional solution, making measurements with the Al_k X-rays feasible. The relative ferrous-to-ferric conversions as a function of dose were similar for the two γ-ray energies, yielding G-values of 1·62 and 1·59 µmol J^?1 for the ⁶⁰Co and ¹³⁷Cs radiations, respectively. The α-particle G-value was 0·52 µmol J^?1, or 31 per cent of that for the ⁶⁰Co γ-rays, in good agreement with previous measurements. The Al_k X-rays had a G-value of 0·92 µmol J^?1 or 57 per cent of that of the ⁶⁰Co radiation. This G-value for the 1·5 keV X-rays is within 20 per cent of the values predicted by current theories, and theoretical values are within the error range of our measurement. The consistency between the experimental value reported here and theoretical G-values for ultrasoft X-rays should be valuable for models of radiation action on biological systems.     

Diurnal Rhythmicity in the Sensitivity of Haemopoietic Cells to Whole-body Irradiation of Mice

Summary

When mice are maintained on a light-dark cycle in which the light begins at 06.00 and ends at 18.00 hours, endogenous spleen colony-forming cells are most radiosensitive at 02.00 and most radioresistant at 22.00 hours. There are three cycles of radiosensitivity with a cycle time of 8 hours. The results are discussed on the basis of the cellular kinetics of the haemopoietic cell system.     

The Influence of Oxygen on the Induction of Radiation Damage in DNA in Mammalian Cells after Sensitization by Intracellular Glutathione Depletion

Summary

Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and non-protein-bound SH (NPSH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10–20 per cent for the three types of radiation damage. Also under severely hypoxic conditions (0·01 μm oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0–30 per cent). At somewhat higher concentrations of oxygen (0·5–10 μm) however, the sensitization amounted to about 90 per cent for the induction of ssb and dsb and about 50 per cent for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen.     

The Effect of 238Pu α-particles on the Mouse Fibroblast Cell Line C3H 10T1/2: Characterization of Source and RBE for Cell Survival

Summary

Considerable interest has been aroused in recent years by reports that the transforming and carcinogenic effectiveness of low doses of high LET radiations can be increased by reducing the dose rate, especially for transformation of 10T1/2 cells in vitro by fission-spectrum neutrons. We report on conditions which have been established for irradiation of 10T1/2 cells with high LET monoenergetic α-particles (energy of 3·2 MeV, LET of 124 keV μm^?1) from ²³⁸Pu. The α-particle irradiator allows convenient irradiation of multiple dishes of cells at selectable high or low dose rates and temperatures. The survival curves of irradiated cells showed that the mean lethal dose of α-particles was 0·6 Gy and corresponded to an RBE, at high dose rates, of 7·9 at 80 per cent survival and 4·6 at 5 per cent survival, relative to ⁶⁰Co γ-rays. The mean areas of the 10T1/2 nuclei, perpendicular to the incident α-particles, was measured as 201 μm², from which it follows that, on average, only one in six of the α-particle traversals through a cell nucleus is lethal. Under the well-characterized conditions of these experiments the event frequency of α-particle traversals through cell nuclei is 9·8 Gy^?1.     

Effect of Free Radicals Induced by Ultrasonic Cavitation on Cell Killing

Summary

The present study was undertaken to elucidate the mechanism of in vitro cell killing induced by 1·0 MHz continuous wave ultrasound at an intensity of 5·8 W/cm². The chemical effects and mechanical effects arising from acoustic cavitation were determined by the amount of liberated iodine and the number of DNA double-strand breaks, respectively. The survival of mouse L cells immediately after irradiation was estimated by counting the number of cells which are not stained by trypan blue and the clonogenicity of surviving cells remaining immediately after irradiation was monitored by colony-forming ability. The effectiveness of the dissolved gases in liberating iodine was in the order O₂ > Ar > N₂ > N₂O ～ 0. However, the effect of dissolved gases on the yield of double-strand breaks of DNA and on the two kinds of end points of cell killing was in the order O₂ = Ar = N₂ > N₂O ～ 0. These results suggest that the different amounts of free radicals induced by ultrasound are not directly related to the ultrasonically induced cell killing. The presence of cysteamine (2 mmol dm^?3) during sonication completely inhibited a decrease in clonogenicity of surviving cells, but did not inhibit that of cell survival immediately after sonication. These results suggest that the decrease of survival immediately after sonication is due to mechanical shear stress arising from cavitation, while the decrease of clonogenicity of the remaining surviving cells is due to free radicals induced by cavitation. The contribution of free radicals to total cell killing was estimated as about 1 per cent at the level of 95 per cent cell killing immediately after sonication.     

Low-dose hypersensitivity after fractionated low-dose irradiation in vitro

Purpose : It was demonstrated previously that some radioresistant tumour cell lines respond to decreasing single, low radiation doses by becoming increasingly radiosensitive. This paper reports the response of four radioresistant human glioma cell lines to multiple low-dose radiation exposures given at various intervals. Three of the cell lines (T98G, U87, A7) were proven already to show low-dose hyper-radiosensitivity (HRS) after single low doses; the fourth, U373, does not show HRS after acute doses. Materials and methods : Clonogenic cell-survival measurements were made in vitro using the Dynamic Microscopic Image Processing Scanner (DMIPS) or Cell Sorter (CS) following exposure to 240kVp X-rays one or more times. Results : A consistent, time-dependent hypersensitive response to a second, or subsequent, dose was observed in the cell lines that demonstrated HRS. This time-dependent change in radiosensitivity did not occur in the radioresistant cell line that did not show HRS (U373). In one cell line that demonstrated strong HRS, T98G, a similar time-dependent hypersensitive response was also seen when the cells were irradiated whilst held in the G1-phase of the cell cycle. In this same cell line, significantly increased cell kill was demonstrated when three very low doses (0.4 Gy) were given per day, 4 h apart, for 5 days, compared with the same total dose given as once-daily 1.2Gy fractions. Conclusions : These data demonstrate the possibility that a multipledose per day, low-dose per fraction regimen, termed 'ultrafractionation', could produce increased tumour cell kill in radioresistant tumours compared with the same total dose given as conventional-sized 2 Gy fractions.     

The Effect of Metabolism on the Transport of 15O-labelled Oxygen through Vicia Faba Roots

The transport of oxygen through continuous gas spaces in the root of the seedling of Vicia faba was investigated, using coincidence-counting of labelled nitrogen-oxygen gas mixtures containing 19 per cent and 0·4 per cent oxygen. The observations indicate that, in diffusing to the elongating zone and the radiosensitive root meristem, a little oxygen is lost to the tissue. This loss occurs in the region of the cotyledon and hypocotyl.     

Metabolic Behaviour of 137Cs-137mBa in the Lactating Goat

Summary

Investigations on the metabolism of ¹³⁷Cs in the lactating goat demonstrated that: (a) the biological half-time for removal of ¹³⁷Cs from the body was about 2–3 days, (b) at steady state, about 7 per cent of the daily dose of ingested ¹³⁷Cs was secreted daily in milk, about 40 per cent in the urine and about 40 per cent in the faeces, (c) the ¹³⁷Cs/K ratio in milk was 1·4 based on a dietary ¹³⁷Cs/K ratio of unity, (d) the mammary gland did not differentiate between ¹³⁷Cs and K, (e) the organ of highest concentration was kidney, with values 1·5 times that of muscle and other soft tissues, (f) the ^137mBa daughter metabolically dissociated from its ¹³⁷Cs parent in vivo so that the gamma activity of the whole blood of the living animal was 5·5 times that as usually determined. These and other findings are discussed and reference made to differences in ¹³⁷Cs metabolism between ruminants and non-ruminants.     

Cytogenetic Effects of Microwave Irradiation on Male Germ Cells of the Mouse

Summary

Hybrid male mice were exposed to 2·45 GHz microwaves for 30 min/day, 6 days a week for two consecutive weeks at power densities of 1·0, 100 or 400 W m^?2, with sham-exposed controls. Rectal temperatures before and after exposure were measured on days 1, 6 and 12. Measurements made on day 1 were treated with caution because of heterogeneity in rectal temperatures taken before exposure between the groups of mice given different treatments. On days 6 and 12, rectal temperatures rose by approximately 1°C in mice sham exposed, or exposed to 1 W m^?2 or 100 W m^?2. Only in the group of mice exposed to 400 W m^?2 was the mean rise in rectal temperature during exposure (about 3°C) significantly increased above the sham value. In groups killed 2–3 days after treatment (mainly meiotic exposure) frequencies of chromosome aberrations in spermatocytes showed no significant heterogeneity although the highest frequency of 1·5 per cent was at the highest (400 W m^?2 power density. Another group killed 30 days after 100 W m^?2 exposures (spermatogonial sampling) showed no significant increase over controls in chromosome aberration frequency. There was a small but significant increase in sperm count with increasing power density in mice killed 12–13 days after exposure, but a non-significant one in those exposed as spermatogonia (killed 41 days later). Thus effects were markedly less severe than those reported previously by Manikowska-Czerska et al. (1985) with a very similar radiation regime and were probably caused by the temperature enhancement.     

Dcr3 inhibit p53-dependent apoptosis in γ-irradiated lung cancer cells

Purpose:?To identify genes responsible for the radiosensitivity, we investigated the role of the differential gene expression profiles by comparing radioresistant H1299 with radiosensitive H460 lung cancer cell lines.

Materials and methods:?mRNA profiles of lung cancer cell lines were assessed using microarray, and subsequent validation was performed with qRT-PCR (Quantitative real time-polymerase chain reaction). The expression levels of differentially expressed genes were determined by Western blot and the radioresistance of lung cancer cell lines was measured by clonogenic assay.

Results:?From the differentially expressed apoptosis-related genes between H1299 and H460, we found Dcr3 (Decoy receptor 3, also known as TNFRSF6B; Tumour necrosis factor receptor super family member 6B) expression was significantly (P?=?4.38 × 10^?7) higher in H1299 cells than H460 cells. Moreover, the Dcr3 mRNA expression level in the radioresistant cell lines (H1299, A549, DLD1, MB231, MB157) was increased in comparison to the radiosensitive cell lines (ME180, Caski, U87MG, MCF7, H460). Overexpression of Dcr3 increased the survival rate of radiosensitive H460, MCF7, and U87MG cells, and knockdown of Dcr3 abolished the radioresistance of A549 cells. The survival rate of p53 (Tumour protein 53)-deficient H1299 after gamma-irradiation was not affected by the suppression of Dcr3 expression. However, when we introduced p53 into H1299 cells, siDcr3 (siRNA of Dcr3) suppressed the radioresistance of H1299 cells by inducing p53-dependent Fas (Fas receptor, also known as TNFRSF6; Tumour necrosis factor receptor super family member 6)-mediated apoptosis pathway.

Conclusion:?Characterisation of gene expression profiles in two lung cancer cell lines revealed that Dcr3 expression and p53-dependent apoptosis signalling pathway regulate cellular response to ionising radiation.     

Radioprotective Effect of an Acute Non-specific Inflammation in Mice

Summary

Protection against 8·7 Gy whole-body γ-irradiation (lethal in 100 per cent of mice by 30 days) was observed in 90 per cent of mice bearing a one-day-old granuloma induced by polyacrylamide beads. When the inflammatory reaction was induced sooner or later a lower or null protection was obtained. A dose-effect relationship between the volume of injected beads and resulting radioprotection was established. The radioprotective effect depends only on the acute non-specific inflammation since hydrocortisone acetate injected into mice before the beads abolished this protection. This inflammatory pattern led to a dose reduction factor of 1·36 ± 0·08 (P < 0·05) for LD 50/30. A 90 per cent survival was observed in mice bearing a one-day-old granuloma when they were injected 1 h before 10 Gy with the granuloma acellular eluate (P < 0·02 compared to a 50 per cent survival observed with polyacrylamide beads alone). Substances with a molecular weight higher than 300 000 are involved in the synergistic radioprotective effect of the granuloma-eluate association.