J.J. Broerse

Mortality of Monkeys after Exposure to Fission Neutrons and the Effect of Autologous Bone Marrow Transplantation

In order to assess the risk of exposure to ionizing radiation in man, and to evaluate the results of therapeutic measures, the mortality of rhesus monkeys irradiated with X-rays and fission neutrons and the effect of autologous bone marrow transplantation have been investigated. The LD50/30d values for X- and neutron-irradiated monkeys amount to 525 and 260 rad respectively, resulting in an r.b.e. of approximately 2 for the occurrence of the bone marrow syndrome. Protection of the animals by autologous bone marrow transplantation was observed up to doses of 860 rad of X-rays and 440 rad of fission neutrons. After both fission-neutron irradiation and X-irradiation in the lowest range of lethal doses, the bone marrow syndrome was found to occur without the concurrent incidence of the intestinal syndrome. The studies indicate that, for humans accidentally exposed to what would otherwise be lethal doses of fast neutrons, bone marrow transplantation may be beneficial.

Radiation Carcinogenesis in Experimental Animals and Its Implications for Radiation Protection

Cancer induction is generally considered to be the most important somatic effect of low doses of ionizing radiation. It is therefore of great concern to assess the quantitative cancer risk of exposure to radiations of different quality and to obtain information on the dose-response relationships for carcinogenesis. Tissues in the human with a high sensitivity for cancer induction include the bone marrow, the lung, the thyroid and the breast in women. If the revised dosimetry estimates for the Japanese survivors of the atomic bomb explosions are correct, there is no useful data base left to derive r.b.e. values for human carcinogenesis. As a consequence, it will be necessary to rely on results obtained in biological systems, including experimental animals, for these estimates. With respect to radiation protection, the following aspects of experimental studies on radiation carcinogenesis are of relevance: Assessment of the nature of dose-response relationships. Determination of the relative biological effectiveness of radiations of different quality. Effects of fractionation or protraction of the dose on tumour development. For the analysis of tumour data in animals, specific approaches have to be applied which correct for competing risks. These methods include actuarial estimates, non-parametric models and analytical models. The dose-response curves for radiation-induced cancers in different tissues vary in shape. This is exemplified by studies on myeloid leukaemia in mice and mammary neoplasms in different rat strains. The results on radiation carcinogenesis in animal models clearly indicate that the highest r.b.e. values are observed for neutrons with energies between 0.5 and 1 MeV. On the basis of such results it might be concluded that the maximum quality factor of 10 for neutrons should be increased. Based on current evidence, an increase by a factor of 2 to 3 seems more realistic than a tenfold rise. The diversity of dose-response relationships point to different mechanisms involved in the induction of different tumours in various species and even in different strains of the same species.

Radiation carcinogenesis in experimental animals

Exposure of man to relatively high doses of ionizing radiation is generally restricted to accidental situations, with very limited knowledge about the actual doses received. Animal experiments can be performed under standardized and controlled conditions and can provide information on the dose-response relationships for radiation carcinogenesis. The risk of inducing neoplastic late effects after total-body irradiation with relatively high doses has been demonstrated for larger animals, such as monkeys and dogs. The bone marrow, the mammary glands and the lungs are among the tissues with the highest susceptibility for radiation carcinogenesis. Experimental results on tumour induction in rodents are summarized with emphasis on the effectiveness in dependence on radiation quality and fractionation or dose rate.

Mammary Carcinogenesis in Different Rat Strains after Irradiation and Hormone Administration

Radiation carcinogenesis of the rat mammary gland was investigated with the objective of investigating the combined effect of oestrogen administration and irradiation. Three rat strains, Sprague-Dawley, Wistar WAG/Rij and Brown Norway, with different susceptibilities to the induction of mammary cancer, have been irradiated with X-rays and mono-energetic neutrons. Increased hormone levels were obtained by subcutaneous implantation of pellets with oestradiol-17 beta (E2). The tumour incidence results were corrected for competing risks and were analysed with a continuous failure time distribution. The latency period for the hormone-treated animals is considerably shorter than for animals with normal endocrinological levels. Administration of the hormone results in an appreciable increase in the proportion of rats with malignant tumours. At the level of hormone administration applied in this study, radiation and hormones appear to produce an additive effect. The effect of hormone administration and irradiation for mammary tumourigenesis is equal for hormone administration one week prior to, or 12 weeks after irradiation. The RBE values for induction of mammary carcinomas after irradiation with 0.5 MeV neutrons have a maximum value of 20 and are not strongly dependent on the hormone levels.

Relative Biological Effectiveness for Neutron Carcinogenesis in Monkeys and Rats

The risks of total-body irradiation with large doses of X rays (average dose 6.7 Gy) and fission neutrons (average dose 3.4 Gy) were investigated by keeping a group of long-term surviving monkeys from an experiment on acute effects under continuous observation. On the basis of the number of animals developing tumors in each group as a function of the total observation period and the average absorbed dose, relative biological effectiveness (RBE) values between 4 and 5 have been derived at these high dose levels. In experiments on mammary carcinogenesis in rats the highest RBE values are observed for neutrons with energies of 0.43 to 1 MeV as produced by the p + T reaction or by the fission process. Based upon linear dose-response curves for neutrons and X rays, a maximum RBE value of 15 was observed for induction of adenocarcinomas in WAG/Rij rats. Appreciably higher RBE values would be obtained if the results of the gamma-ray exposure, indicating a nearly quadratic dose-response relationship, were used as a baseline. For radiation protection applications it should be realized, however, that such an increase will be caused by the lower efficiency of low-linear-energy-transfer radiation rather than by an increase in efficiency of the neutron irradiation at low doses.

Prolactin concentration in plasma and susceptibility to mammary tumors in female rats from different strains treated chronically with estradiol-17β

SummaryProlactin is associated with the development of mammary tumors in rats. The aim of the present study was to evaluate whether strain differences in susceptibility to the development of mammary tumors could be explained by genetic differences in the response of the pituitary to chronic stimulation by estrogens. Prolactin levels were measured in plasma from rats of the Sprague-Dawley, Wistar WAG/Rij and Brown Norway BN/BiRij strains before and at different times after subcutaneous implantation of estradiol-17β in cholesterol/paraffin pellets. In all strains plasma prolactin was elevated from the second week after implantation of the pellet, although there were quantitative differences between the responses. At 32 weeks after implantation of the pellets the plasma level of prolactin in Sprague-Dawley rats was 1247 ± 367 ng NIAMDD prolactin RP-1/ml (mean ± S.E.M), whereas Wistar WAG/Rij and Brown Norway BN/BiRij had plasma prolactin levels of 679 ± 211 and 182 ± 19 ng/ml respectively. Between 52 and 104 weeks after implantation these values rose to 4016 ± 1116, 5004 ± 1053 and 808 ± 129 ng/ml respectively. The plasma concentration of prolactin of rats in this age group was strongly associated with the occurrence of pituitary adenomas in all three strains. In untreated rats, the concentration of prolactin in the plasma increased with age to only 200–400 ng/ml at 12–24 months of age but no significant differences were observed between the three rat strains. It is concluded that observed differences in spontaneous and estrogen-mediated mammary tumor development in these rat strains cannot be explained by genetic differences in the plasma concentration of prolactin. The development of malignant mammary tumors after estrogen treatment appears to be associated with the extent of the increase in plasma prolactin induced by the estrogen.

Long-term effects of total-body irradiation on the kidney of Rhesus monkeys

PURPOSE To investigate the long-term effects of total-body irradiation (TBI) on kidneys in non-human primates. METHODS AND MATERIALS The kidneys of Rhesus monkeys were histologically examined at 6-8 years after TBI with low single doses of 4.5-8.5Gy or two fractions of 5.4Gy. The kidneys of age-matched non-irradiated monkeys served as controls. Irradiation was performed on adult monkeys aged about 3 years; 6-8 years later animals were sacrificed and the kidneys removed and processed for histology. A semi-quantitative scoring system was used to evaluate overall histological damage. Glomerular changes were also morphometrically analysed according to previously published criteria. In selected dose groups (pro)thrombotic and inflammatory changes were investigated by immunostaining cryosections with antibodies against von Willebrand factor (vWF), leukocytes and macrophages. RESULTS Histological changes were generally mild and only seen in kidneys irradiated with doses higher than 7 Gy. Glomerular changes were characterized by increased mesangial matrix and capillary dilatation. Tubulo-interstitial changes included hypercellularity, fibrosis and mild tubular atrophy. The mean glomerular area expressing vWF protein in the irradiated kidneys was not different from that in the age-matched controls. Numbers of infiltrating leukocytes were not significantly different between irradiated kidneys and controls. However, slightly increased numbers of macrophages were present in the renal cortex after irradiation. CONCLUSIONS Renal damage after TBI of Rhesus monkeys with single doses of 4.5-8.5 Gy or two fractions of 5.4 Gy was mild, even after follow-up times of 6-8 years.Purpose : To investigate the long-term effects of total-body irradiation (TBI) on kidneys in non-human primates. Methods and materials : The kidneys of Rhesus monkeys were histologically examined at 6–8 years after TBI with low single doses of 4.5–8.5 Gy or two fractions of 5.4 Gy. The kidneys of age-matched non-irradiated monkeys served as controls. Irradiation was performed on adult monkeys aged about 3 years; 6–8 years later animals were sacrificed and the kidneys removed and processed for histology. A semi-quantitative scoring system was used to evaluate overall histological damage. Glomerular changes were also morphometrically analysed according to previously published criteria. In selected dose groups (pro)thrombotic and inflammatory changes were investigated by immunostaining cryosections with antibodies against von Willebrand factor (vWF), leukocytes and macrophages. Results : Histological changes were generally mild and only seen in kidneys irradiated with doses higher than 7 Gy. Glomerular changes were characterized by increased mesangial matrix and capillary dilatation. Tubulo-interstitial changes included hypercellularity, fibrosis and mild tubular atrophy. The mean glomerular area expressing vWF protein in the irradiated kidneys was not different from that in the age-matched controls. Numbers of infiltrating leukocytes were not significantly different between irradiated kidneys and controls. However, slightly increased numbers of macrophages were present in the renal cortex after irradiation. Conclusions : Renal damage after TBI of Rhesus monkeys with single doses of 4.5–8.5Gy or two fractions of 5.4Gy was mild, even after follow-up times of 6–8 years.

Tumorigenesis in High-Dose Total Body Irradiated Rhesus Monkeys—A Life Span Study

In the early sixties, studies have been performed at the TNO-Institutes for Health Research on acute effects of high dose total body irradiation (TBI) with X-rays and fission neutrons in Rhesus monkeys and the protective effect of autologous bone marrow transplantation (BMT). The surviving animals of this study were kept to investigate late radiation effects, ie, tumorigenesis. TBI in combination with chemotherapy, followed by rescue with BMT is increasingly used for the treatment of hematological malignancies and refractory autoimmune disease. The risk of radiation carcinogenesis after this treatment is of growing concern in man. Studies on tumor induction in nonhuman primates are of relevance in this context since the response of this species to radiation does not differ much from that in man. The group of long-term surviving monkeys comprised nine neutron irradiated animals (average total body dose 3.4 Gy, range 2.3—4.4 Gy) and 20 X-irradiated monkeys (average total body dose 7.1 Gy, range 2.8—8.6 Gy). A number of 21 age-matched nonirradiated Rhesus monkeys served as a control-group. All animals were regularly screened for the occurrence of tumors. Complete necropsies were performed after natural death or euthanasia. At postirradiation intervals of 4—21 years an appreciable number of malignant tumors was observed. In the neutron irradiated group eight out of nine animals died with 1 or more malignant tumors. In the X-irradiated group this fraction was 10 out of 20. The tumors in the control group, in seven out of 21 animals, appeared at much older age compared with those in the irradiated cohorts. The histogenesis of the malignant tumors was diverse, as was the case for benign tumors. The observed shortening of latency periods and life span, as well as, the increase of mean number of tumors per tumor bearing animal for benign neoplasms parallels the trend observed for malignant tumors. The results of this study were compared to other radiation late effects after TBI followed by different BMT treatment modalities in Rhesus monkeys. The observation that the carcinogenic risk of TBI in the Rhesus monkeys is similar to that derived from the studies of the Japanese atomic bomb survivors and the increase of the risk by a factor of 8 emphasizes the need for regular screening for secondary radiation-induced tumors in long-term surviving patients after TBI followed by BMT.

Induction of mammary tumors in rats by single-dose gamma irradiation at different ages.

The effect of age at exposure on induction of mammary tumors was studied in female rats of the inbred WAG/Rij strain. Groups of 40 animals were exposed to a single total-body dose of 1 or 2 Gy of 137Cs gamma radiation at ages of 8, 12, 16, 22, 36 or 64 weeks and were observed for life. Mammary tumors, identified as nodules persisting and growing for 6 weeks, were resected and classified histologically as carcinoma or fibroadenoma. The age-specific incidence of mammary carcinoma was compared with that in a group of 120 unirradiated control rats, using lifetime statistical analysis with both parametric and nonparametric methods. The excess normalized risk of carcinoma was 0.9 for 1 Gy and 2.2 for 2 Gy in age groups 8-36 weeks, with no significant differences between the age groups. However, irradiation at 64 weeks yielded fewer carcinomas than in the controls, the excess normalized risk being -0.7 and -0.3 for 1 and 2 Gy, respectively. The occurrence of one or more fibroadenomas did not influence the incidence of carcinoma. The present data agree closely with the results reported previously for rats irradiated at age 8 or 17 weeks with a dose of 1.2 Gy. The reduced risk of radiation exposure at midlife is consistent with the available epidemiological data for exposed women. Although our findings have been obtained with a single total-body dose that is several orders of magnitude higher than the multiple doses delivered to the mammary gland during mammography, it is suggested that radiological screening for mammary cancer after the age of menopause will not increase the normal incidence of breast cancer.

The carcinogenic risk of high dose total body irradiation in non-human primates

PURPOSE High dose total body irradiation (TBI) in combination with chemotherapy, followed by rescue with bone marrow transplantation (BMT), is increasingly used for the treatment of haematological malignancies. With the increasing success of this treatment and its current introduction for treating refractory autoimmune diseases the risk of radiation carcinogenesis is of growing concern. Studies on tumour induction in non-human primates are of relevance in this context since the response of this species to radiation does not differ much from that in man. MATERIALS AND METHODS Since the early sixties, studies have been performed on acute effects in Rhesus monkeys and the protective action of bone marrow transplantation after irradiation with X-rays (average total body dose 6.8 Gy) and fission neutrons (average dose 3.4 Gy). Of those monkeys, which were irradiated and reconstituted with autologous bone marrow, 20 animals in the X-irradiated group and nine animals in the neutron group survived more than 3 years. A group of 21 non-irradiated Rhesus monkeys of a comparable age distribution served as controls. All animals were regularly screened for the occurrence of neoplasms. Complete necropsies were performed after natural death or euthanasia. RESULTS At post-irradiation intervals of 4-21 years an appreciable number of tumours was observed. In the neutron irradiated group eight out of nine animals died with one or more malignant tumours. In the X-irradiated group this fraction was 10 out of 20. The tumours in the control group, in seven out of the 21 animals, appeared at much older age compared with those in the irradiated cohorts. The histogenesis of the tumours was diverse with a preponderance of renal carcinoma, sarcomas among which osteosarcomas, and malignant glomus tumours in the irradiated groups. CONCLUSIONS When corrected for competing risks, the carcinogenic risk of TBI in the Rhesus monkeys is similar to that derived from the studies of the Japanese atomic bomb survivors. The increase of the risk by a factor of 8, observed in the monkeys, indicates that patients are likely to develop malignancies more frequently and much earlier in life after TBI than non-exposed individuals. This finding underlines the necessity of regular screening of long-term surviving patients subjected to TBI and BMT.