Long-term expression of transforming growth factor TGF beta 1 in mouse skin after localized beta-irradiation.

The long-term expression of TGF beta 1 in mouse skin after localized irradiation with a beta-emitting source is reported. The skin of CBA/ca mice was exposed to 50 Gy superficial beta radiation from an 11 mm strontium-90 source. Such a dose produced an acute moist desquamation reaction in 100% of the animals, which was macroscopically resolved within 30 days. The acute response was followed by progressive remodelling of dermal tissues as characterized by histological means. The expression of TGF beta 1 was followed for 12 months after irradiation and showed three distinct waves of expression at the RNA level. Levels of expression initially rose to 230% above controls at 6 h before returning to control levels around 24-48 h. Expression then rose again to 169 and 234% above controls at 14 and 28 days post-irradiation respectively. Levels then declined to those of the controls by 2 months. A progressive increase in expression was then noted after 3 months, which peaked around 9 months and was resolved by 12 months. In a parallel study the skin of 144 CBA/ca mice was exposed to 50 Gy superficial beta radiation from 2 x 4 cm Thulium-170 source and compared with a similar group of sham-irradiated controls. The irradiated group showed a cumulative tumour incidence of 54.3% compared with 0% incidence in the sham-irradiated group. Of the 45 radiation-induced tumours a representative sample of 16 (nine malignant fibrous histiocytomas; three fibrosarcomas; two fibromas; one squamous cell carcinoma; one rhabdomyosarcoma) were selected for further study. Semiquantitative PCR on all these tumours showed elevated levels of TGF beta 1 expression ranging from 1.8 to 87-fold above the levels found in normal skin. This study is part of ongoing investigations into the long-term effects of single accidental exposures. The 50 Gy dose used is comparable with the surface doses obtained by some of the victims of the Chernobyl accident.

Expression of transforming growth factor-beta 1 in mouse skin during the acute phase of radiation damage.

Transforming growth factor-beta (TGF beta 1) plays a central role in wound healing, so its perturbation by radiation may contribute to the acute and late effects seen in irradiated skin. TGF beta 1 mRNA expression was measured by PCR, in the skin of the CD1 and CBA mouse, exposed to Sr-90 beta from an 11-mm diameter source. TGF beta 1 mRNA expression increased sharply after doses between 1 and 10 Gy and plateaued at approximately 200% above controls after doses between 20 and 50 Gy. Immunohistochemistry showed that the TGF beta 1 protein was confined to the dermis and suprabasal cells with none in basal cells. A dose of 50 Gy produces an acute desquamative reaction in 100% of mice that is resolved in 30 days. After the same dose, TGF beta 1 mRNA expression fell below the controls at 3 h (-9.4% in the CD1 and -44% in the CBA mouse); rose sharply at 6-12 h (+124% CD1, +230% CBA), returned to control levels by 24-48 h, then rose progressively to approximately 200% above the controls between days 7 and 14. TGF beta 1 mRNA expression remained elevated at 100-200% above controls until the end of the experiment at 55 days. The significance of these changes in TGF beta 1 is discussed in the context of the early stress response reaction to radiation, the acute inflammatory and the later chronic fibrosis of the skin.

The effect of whole-body gamma-irradiation on localized beta-irradiation-induced skin reactions in mice.

The combined effects of whole-body radiation and localized radiation trauma have received scant experimental attention. However, in the recent accidents at Chernobyl and Goiania skin damage from beta-contamination was combined with total-body radiation and in many cases the skin lesions which covered large surfaces of the body were severe and recovery was prolonged. This paper models the immunosuppressive effects of whole-body gamma-radiation in the sublethal to lethal range (1-11 Gy) on the skin reactions produced by 50 Gy of superficial beta-radiation. For gamma ray doses < 4 Gy no synergistic effects were detectable. For gamma-ray doses of 4, 6 and 8 Gy there was a 4-5-day prolongation in time-course of the skin reaction but no significant exacerbation of its severity. The overall time for the resolution of the skin reaction (45 days) was also unaffected by the relatively high whole-body doses. These rather surprising findings of minimal synergy between whole-body exposure and a localized severe beta burn to the skin are perhaps explained by the mismatch between the maximal immunosuppression at 2-10 days postirradiation and the timing of the skin damage at 10-25 days.

Differences in vascular response between primary and transplanted tumours.

The vast majority of studies on tumour vasculature are performed on transplanted tumours in rodents. However, it is known that there may be differences between primary and transplanted lesions. The purpose of this study is to test whether a specific vascular response is similar in primary tumours and in transplanted tumours derived from them. The technique used was to give an intraperitoneal injection of 5 mg kg-1 hydralazine, which is known to result in hypoxia in transplanted tumours. Changes in perfusion were indicated by changes in metabolism, monitored using 31P Magnetic Resonance Spectroscopy. The primary tumours were induced by local irradiation many months previously and only 4/11 (36%) of these responded to hydralazine. One of the non responders was subsequently transplanted into isogeneic mice to produce a tumour line which was histologically very similar to the primary. Of these 16/17 (94%) responded. The difference is statistically significant (P = 0.001). The reasons for this difference are not known. A number of possibilities are discussed and in the authors' opinion, the most likely cause is that it results from an artefact of transplantation.

Experimental studies of radiation carcinogenesis in the skin: a review.

Skin has been widely used in radiation carcinogenesis studies because of the accessibility and visibility of its tumours. Both rat and mouse models have proved to be sensitive, reproducible systems to study the dose and time response of cancer induction following different modes and qualities of radiation exposure. This paper discusses the variation in the shape of the low-LET dose responses from purely linear with no threshold to the highly quadratic curves with significant thresholds, although a linear response is more consistently reported following high-LET radiations. Some dose-response curves show no tendency to turnover at high doses, others show a declining incidence of skin cancer at the highest doses. Protraction or fractionation of the dose reduces the carcinogenic effect in rat skin, whilst the reported dose rate studies in mice are equivocal regarding any sparing effect. Mouse skin cancer studies, in particular, have empirically refuted the 'hot particle hypothesis'. The extensive studies of Albert and Burns highlight hair follicle damage at 300 microns depth as critical in the development of the majority of rat skin tumours. In contrast, mouse studies report a wide variety of cell types as the putative 'cells at risk' in the skin from the spectrum of epidermal and dermal tumours which are induced, and which have been found to be amenable to classification using human pathological categories. Despite these interspecies differences, it is shown that all of the experimental data for radiogenic skin cancer, when expressed per unit area of skin, fall on a relatively narrow and well defined response curve, which is approximately two orders of magnitude more sensitive than the human skin cancer dose response.

Skin carcinogenesis following uniform and nonuniform beta irradiation.

In practical situations where workers or the general public may be exposed to ionizing radiation, the resulting irradiation is rarely uniform. The risk figures and dose limits recommended by the International Commission on Radiological Protection (ICRP) are based largely on clinical and epidemiological studies of reasonably uniform irradiated organs. The paucity of clinical or experimental data for highly nonuniform exposures has prevented the ICRP from providing adequate recommendations applicable to this practical situation. This weakness has led on a number of occasions to the postulate that highly nonuniform exposures of organs, such as the lung or the skin, could be 100,000 times more carcinogenic than ICRP risk figures would predict. This so-called "hot-particle hypothesis" found little support among reputable radiobiologists, but could not be clearly and definitively refuted on the basis of experiment. An experiment design, based on skin tumour induction in mouse skin, is described which was developed to test the "hot-particle hypothesis." In collaboration with the Radiobiology Department of St. Bartholomew's Hospital Medical College, London, the skin of 1200 SAS/4 male mice has been exposed to a range of uniform and nonuniform sources of the beta emitter 170Th (Emax 1 MeV). Nonuniform exposures were produced using arrays of 32 or 8 2-mm-diameter sources distributed over the same 8-cm2 area as a uniform control source. Average skin doses varied from 2-100 Gy. The results for the nonuniform sources show a 30% reduction in tumour incidence by the 32-point array at the lower mean doses compared with the response from uniform sources. The eight-point array showed an order-of-magnitude reduction in tumour incidence compared to uniform irradiation at low doses. These results, in direct contradiction to the "hot-particle hypothesis," indicate that nonuniform exposures produce significantly fewer tumours than uniform exposures.

Lung tumour induction in mice after X-rays and neutrons.

Dose-response curves were determined for pulmonary adenomas and adenocarcinomas in mice after single acute doses of 200 kVp X-rays and cyclotron neutrons (E = 7.5 MeV). A serial-killing experiment established that the radiation induces the tumours and does not merely accelerate the appearance of spontanoeus cancers [corrected]. The dose versus incidence (I) of tumours in male and female mice for X-ray doses between 0.25 and 7.5 Gy is 'bell-shaped' and best fitted with a purely quadratic induction and exponential inactivation terms, i.e. I = A + BD2e-alpha D. In contrast, the tumour dose-response after 0.1-4.0 Gy of neutrons is best fitted by I = A + BDe-alpha D and is steeply linear less than or equal to 1 Gy, peaks between 1 and 3 Gy and sharply declines at 4.0 Gy. The data for the female mice less than or equal to 1 Gy neutrons are best fitted to the square root of the dose. A major objective of the experiments was to derive neutron RBE values. Because of the differences between the X-ray (quadratic) and neutron (linear) curves, the RBEn will vary inversely with decreasing X-ray dose. The RBE values at 1 Gy of X-rays derived from the B coefficients in the above equations are 7.4 +/- 3.2 (male and female); 8.6 +/- 3.6 (female) and 4.7 +/- 1.8 (male). These are high values and imply even higher values at the doses of interest to radiation protection. If, however, one restricts the analysis to the initial, induction side of the response (less than or equal to 1 Gy neutrons, less than or equal to 3 Gy X-rays) then good linear fits are obtainable for both radiations and indicate neutron RBE values of 7.4 +/- 2.3 for female mice and 4.5 +/- 1.8 for males, and these are independent of dose level.

The radiosensitivity of embryos of domestic chickens and black-headed gulls.

Eggs of domestic chickens and black-headed gulls were continuously exposed to gamma-rays during incubation, using dose rates ranging from 0.004 to 0.08 Gy h-1 for 20 days. Acute-dose experiments were also conducted, and eggs were irradiated on day 10 of incubation with doses of between 1.92 and 28.8 Gy. Hatchability and numbers reaching full-term developed were affected only after chronic doses of 9.6 Gy and acute doses of 4.8 Gy or higher. Maximum embryo mortality occurred around days 10-11 of incubation and just before hatching, in all experiments. An increase in foot and limb deformities was observed above acute and chronic doses of 9.6 Gy.

Proliferation of type II pneumonocytes after X-irradiation.

This paper reports preliminary data on the proliferative response of type II cells in the mouse lung over a five-month period after external thoracic doses of 2, 5, 10 and 12 Gy of X-rays. The DNA labelling index (LI) of control (0 Gy) mice was at all times exceedingly low (0.3-0.4 per cent). The LI after 2 and 5 Gy showed a slight though transient fall below controls during the first week post-irradiation, and thereafter the LIs were similar to the controls for the 5 months of the experiment. The LI after 10 and 12 Gy again showed a significant depression during the first week, but this was followed by a significant increase (P = 0.01) in LI which peaked at 4 weeks after irradiation. The LI returned to control values at 3-4 months and again rose significantly (P = 0.05) at 5 months. The first wave of proliferation corresponds to data showing an increase in surfactant in alveolar fluids within 2-6 weeks of 10-15 Gy of X-rays; and the second wave coincides with the pneumonitic phase and is consistent with a delay before the alveolar epithelial continuity is sufficiently compromised by the low rates of type I cell loss to trigger a compensatory wave of type II cell divisions. This relatively chronic radiation response is discussed and contrasted with the dramatic and immediate hyperplastic responses which many toxic irritants produce in type II epithelial cells.

Radiation effects in the lung.

This article outlines the principles of radiobiology that can explain the time of onset, duration, and severity of the complex reactions of the lung to ionizing radiation. These reactions have been assayed biochemically, cell kinetically, physiologically, and pathologically. Clinical and experimental data are used to describe the acute and late reactions of the lung to both external and internal radiation including pneumonitis, fibrosis and carcinogenesis. Acute radiation pneumonitis, which can be fatal, develops in both humans and animals within 6 months of exposure to doses greater than or equal to 8 Gy of low LET radiation. It is divisible into a latent period lasting up to 4 weeks; an exudative phase (3-8 weeks) and with an acute pneumonitic phase between 2 and 6 months. The latter is an inflammatory reaction with intra-alveolar and septal edema accompanied by epithelial and endothelial desquamation. The critical role of type II pneumonocytes is discussed. One favored hypothesis suggests that the primary response of the lung is an increase in microvascular permeability. The plasma proteins overwhelm the lymphatic and other drainage mechanisms and this elicits the secondary response of type II cell hyperplasia. This, in its turn, produces an excess of surfactant that ultimately causes the fall in compliance, abnormal gas exchange values, and even respiratory failure. The inflammatory early reaction may progress to chronic fibrosis. There is much evidence to suggest that pneumonitis is an epithelial reaction and some evidence to suggest that this early damage may not be predictive of late fibrosis. However, despite detailed work on collagen metabolism, the pathogenesis of radiation fibrosis remains unknown. The data on radiation-induced pulmonary cancer, both in man and experimental animals from both external and internal irradiation following the inhalation of both soluble and insoluble alpha and beta emitting radionuclides are reviewed. Emphasis is placed on the data showing that alpha emitters are at least an order of magnitude more hazardous than beta/gamma radiation and on recent data showing that the more homogeneous the irradiation of the lung, the greater is the carcinogenic hazard which contradicts the so-called "hot particle" theory.

Lung tumour induction in mice after uniform and non-uniform external thoracic X-irradiation.

The aim of this study was to investigate the validity of the ICRP procedure of using average tissue/organ dose in estimating carcinogenic risk. It has been suggested that highly non-uniform exposure ('hot spots') is much more carcinogenic than an equivalent dose delivered uniformly. In a series of experiments, mice were irradiated with X-rays either uniformly to the thorax or non-uniformly with 72 1-mm microbeams which irradiated approximately 20 per cent of the total lung volume. Two experiments involving uniform irradiation showed a peaked tumour incidence curve with a maximum at 5 Gy. The first 'microbeam' study also produced a pronounced peak in the dose response with a maximum tumour incidence at 1 Gy average lung dose or 5 Gy to the irradiated lung tissue. This implied the use of average tissue dose might underestimate the carcinogenic hazard of non-uniform exposure. Later, more extensive, microbeam experiments failed to replicate this finding. The results were nearly similar to those for uniform irradiation, with a slight increase in tumour incidence from 2.5-5.0 Gy average lung dose. These results imply that for these irradiation conditions the ICRP dose averaging procedure remains valid.

The influence of the hair cycle on the thickness of mouse skin.

The data on mouse skin thickness reported here was prompted by the need to know the true position of basal cells of the epidermis and hair follicles as these are important "cells at risk" for a variety of skin reactions including carcinogenesis following exposure to radiation. There is little reliable data in the literature and most previous reports have ignored the shrinkage of skin that occurs because of its natural elasticity. The values determined for mouse flank skin in telogen--the resting phase of the hair cycle for the different skin layers--are epidermis 10 micron, corium 250 micron, adipose layer 150 micron, and hair follicle depth 150 micron. Three days after chemical depilation which triggers the hair follicles into active cycle (anagen) the epidermis doubles in thickness, remains at this value for 7 days, and then gradually returns to telogen values by day 18. The corium and adipose layers also increase significantly to reach approximately 390 micron and approximately 260 micron, respectively, by day 10 and then return to control values from day 15 onward. The change in hair follicles depths are more dramatic with active follicle basal cells reaching approximately 450-550 micron into the adipose layer between days 7 and 15. One important finding is that chemical depilation does not affect the telogen thickness of skin-the teleogen values for the epidermis and dermis immediately prior to and immediately after depilation were similar to those 23 days later at the beginning of the next telogen phase.

Changes in the DNA labelling index after beta irradiation of the mouse epidermis.

This study looked at the changes in the interfollicular DNA labelling index (LI) with time after strontium-90/yttrium-90 beta irradiation of approximately 100 mm2 of mouse flank skin, after a dose of 100 Gy which produces transitory moist desquamation. Within 24 hr of such a dose the LI of the irradiated area was essentially zero (0.07 +/- 0.03%), whilst those of the side area and of the control area were 15.0 +/- 2.6% and 21.4 +/- 2.7%, respectively. The LI of the side and the control areas then fell within 3-5 days to approximately 4% and approximately 2% respectively, whilst that of the irradiated area rose rapidly to a peak value of 30.2 +/- 1.7% at 10 days post-irradiation. There was a 20% reduction in the diameter of the area with detectable radiation damage within 5 days, and this is primarily due to cell proliferation and migration from the unirradiated margins of the field. In contrast, between days 10 and 20 the major source of repopulation is probably derived from local migration and proliferation of surviving hair follicle basal cells within the irradiated field.

Nonstochastic effects of different energy beta emitters on the mouse skin.

The effect of irradiating varying areas of mouse skin from 860 down to 0.8 mm2 with different energy beta emitters was studied to clarify protection problems of localized doses to the skin. Both 90Sr and 170Tm sources show area effects for dose-response curves. The 90Sr doses that produced moist desquamation in 50% of irradiated fields (MD-50 doses) were 22, 42, 70, and 1000 Gy for 400-, 95-, 20-, and 0.8-mm2 sources. The MD-50 doses for 170Tm were 50, 54, 90, and 170 Gy for 860-, 64-, 20-, and 3.1-mm2 sources. Thus for the larger 170Tm sources there is much less area effect. There was no significant difference in effect between the different energy 90Sr and 170Tm sources for moist desquamation. A simple hypothesis based upon the repopulation of epithelial cells from the edges of the irradiated field and/or from surviving follicle basal cells can explain these area and energy effects in the mouse and in parallel pig skin experiments. The doses needed for 50% of the mice to show ulceration after 64-, 20-, and 3.1-mm2 170Tm sources were 260, 550, and 8300 Gy, respectively, while those for 90-, 20-, and 0.8-mm2 90Sr sources were 150, 210, and 3100 Gy. Thus there is a definite area and energy effect for these sources for this deep dermal damage. The steep rise in dose needed to produce given skin reactions for the smallest area (0.8 mm2) should reassure those faced with assessing the hazard of sub-millimeter-sized particles in/on human skin.

The proliferation kinetics of pulmonary alveolar macrophages.

In this study the proliferation kinetics of pulmonary alveolar macrophages (PAMs) are determined using [3H]thymidine labeling, flow cytofluorimetry, metaphase arrest, and the percentage of labeled mitoses. The demonstration of in situ PAM proliferation is demonstrated beyond doubt. Comparison of the turnover time, calculated from cell kinetic parameters, with the experimentally determined migration times shows that the contribution proliferating PAMs make to their own population size is significant. Indeed, a strong case may be made to show that some 70% of the PAM population is produced by intra-alveolar divisions of "free" PAMs.

Evidence for the pulmonary origin of alveolar macrophages.

This paper supports the hypothesis that some form of pulmonary alveolar macrophage (PAM) production occurs within the lung in the normal steady state. The study involved monitoring the change in number of labelled PAMs following two modes of irradiation--the first with the thorax being irradiated and the rest of the mouse shielded, the second with the thorax shielded and the body irradiated. Also measurements of monocyte and PAM numbers after a single bone marrow irradiation were carried out. Finally, the labelling indices of monocytes in both control and thorax irradiated mice were measured. Both the number of monocytes and PAMs, along with the labelling indices of monocytes and PAMs after irradiation, indicate the independence of PAMs from a monocyte precursor population, and also provide evidence for a pulmonary origin of PAMs.