Bone sarcoma characteristics and distribution in beagles injected with radium-226.

A total of 155 primary bone sarcomas were found in 131 of the 246 beagles injected with 226Ra and 5 primary bone sarcomas were found in 4 of the 158 unexposed controls. Of these 155 bone sarcomas, 146 (94%) were osteosarcomas and 9 were non-osteosarcomas. An additional 31 primary bone sarcomas (28 osteosarcomas) developed in 44 dogs terminated from the main study because of limb amputation for bone sarcoma. Non-osteosarcomas predominated in both the controls and the second lowest of six logarithmically increasing dose levels (there were no bone sarcomas in the lowest dose group). Osteosarcomas predominated at the higher dose levels, and incidence tended to increase as dose increased. The 146 osteosarcomas were distributed quite evenly between males and females (72:74). Of the 9 non-osteosarcomas, 6 occurred in males and 3 in females. The ratio of bone sarcomas of the appendicular skeleton to those in the axial skeleton was 110:45, with osteosarcomas occurring more often in the appendicular skeleton (108:38). Cases of multiple primary bone sarcomas in dogs injected with 226Ra were found only in the four highest dose groups. Amputations were performed on 44 of the 96 dogs (94 injected and 2 unexposed) that developed appendicular bone sarcomas. A statistical study of the distribution of bone sarcomas among 16 separate bone groups showed a statistically significant correlation to cancellous skeletal surface, but the variability among bone groups was too large for this relationship to be of real predictive value. It is postulated that the distribution of bone sarcomas reflects primarily the relative cell division rates in the bone groups and secondarily the radiation dose distribution, with the highest occurrence of bone sarcoma in the humeri, pelvis, femora and tibiae/fibular tarsal, and no occurrence in the coccygeal vertebrae, sternum, forepaws or hindpaws.

Genotoxicity of the phosphoramidate agent tabun (GA).

Five mutagenicity tests were performed on Agent GA (Tabun, phosphoramidocyanidic acid, dimethyl-, ethyl ester) as part of a program to demilitarize chemical warfare agents. GA was mutagenic in Salmonella spp. assays with S-9 and it was a direct-acting mutagen to mouse lymphoma cells. GA did not promote unscheduled DNA synthesis in rat hepatocytes; it induced sister chromatid exchanges in mouse cells in vitro but in vivo. The conclusion that GA is a weakly acting mutagen is supported by the fact that it was mutagenic in only three of the five assays, and that increases in mutagenicity were often less than 2-fold the controls and occurred near toxic levels.

Bone sarcoma characteristics and distribution in beagles fed strontium-90.

A total of 66 primary bone sarcomas were diagnosed in 47 beagles; 43 of these dogs were part of the 403 beagles fed 90Sr and 4 were part of the 162 controls. Multiple primary bone sarcomas were found in 15 of the 47 beagles (32%). The incidence of multiple primary bone sarcoma was restricted to the two highest dose groups, except for a single control dog which developed two bone sarcomas. A threshold-like radiation dose response was observed; no sarcomas were observed in the lowest three dose groups, but the number of primary bone sarcomas increased rapidly in the higher dose groups. Of the 66 primary sarcomas, 49 were osteosarcomas (74%). As the dose increased, the proportion of osteosarcomas increased sharply, 4/10 (40%), 26/29 (90%), and 16/18 (89%), in the three highest dose groups. Thirteen of the bone sarcomas of other types occurred in males, and 4 in females, whereas 21 osteosarcomas occurred in males, and 28 in females. The ratio of bone sarcomas of the appendicular skeleton to those in the axial skeleton was 40:26, with osteosarcomas occurring more often in the appendicular than the axial skeleton (32:17), whereas nonosteogenic tumors showed no predilection (8:9). A statistical study of the distribution of bone sarcomas among 16 separate bone groups showed a correlation only with the distribution of cancellous bone volume-to-surface ratio and not with either skeletal mass distribution or dose distribution. The highest occurrence of sarcomas was in the humeri, femora, and mandible, and no occurrence in the coccygeal vertebrae, paws, or sternum. It is postulated that the distribution of bone sarcomas reflects a critical combination of the osteosarcoma precursor cell population, their cell division rate, and the radiation dose absorbed by these cells.

The lymphotoxic action of vanadate.

The acute toxicity of ammonium metavanadate (15.5 mg/kg) in mice was investigated to examine the induction of lymphoid necrosis to (1) verify the reproducibility of the lesions in the thymus, lymph nodes, and spleen; (2) determine whether the necrosis of lymphoid tissue previously observed during the first 3 days post-treatment but absent at 14 days was the result of differences in sensitivity of the mice or the result of recovery from the effects of vanadium; and (3) determine whether differences in the presence and the degree of necrosis between thymus and spleen were correlated with differences in the uptake of vanadium in these tissues. A timed sacrificed study was conducted in conjunction with a 48V tracer. In this study, BALB/C mice were injected subcutaneously (s.c.) with ammonium metavanadate solution (15.5 mg/kg). Groups of mice were sacrificed at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 14, 21, and 28 days postexposure. Lymphoid necrosis was found in the thymus, spleen, lymph nodes, and bone marrow, with the necrosis being most severe in the thymus. The necrosis was moderate at 0.5 days, most severe at 2 to 3 days, with recovery beginning at 4 days, and proceeding to full recovery at 14 to 28 days. At 0.5 days post-treatment, the concentration of vanadium in thymus and spleen was 4.4 and 8.3 micrograms/g, respectively. At all post-treatment periods, with the exception of the 1- and the 4-day periods, the concentration of vanadium in spleen was significantly higher than in the thymus, p less than 0.05. The treated animals showed neurological signs (ataxia, convulsion, dyspnea, and paralysis of hind legs) between 5 min and 54 hr post-treatment, but the concentration of vanadium in the brain was very low during this period (less than 5.2% of blood concentration).

Interspecies scaling of risk for radiation-induced bone cancer.

The induction of bone cancer in mice, dogs and humans, due to protracted alpha-irradiation from skeletal burdens of radium, was found to be represented by a single dose-rate/time/response function, when time was normalized with respect to species natural life-span. In the absence of other causes of death, the median time to death from bone cancer after 226Ra intake is given by tm* = 790-d*-0.29, based on the dog data, with -d* the time-weighted average absorbed dose rate in cGy/mLSF to skeleton and where time is measured as milli-life-span-fraction. On the basis of life-span scaling of the time dimension, data on cancer induction from studies with laboratory animals can be scaled to estimate human risks in a three-step process involving a three-dimensional analysis. The overall cancer risk distribution is shown to be a mountain-like surface rising from a Euclidean plane formed by the dose rate and survival time co-ordinates. At lower dose rates the time required for cancer induction may exceed the natural life-span yielding a quasi-threshold for cancer risk. For intakes of 226Ra in young adults this quasi-threshold is predicted to occur at a cumulative life-time alpha-radiation dose to the skeleton of about 1 Gy.

Time and dose-response study of the effects of vanadate on rats: morphological and biochemical changes in organs.

Vanadate at a dosage level of 0.9 mg V/kg per day produced acute toxic signs in rats when injected subcutaneously for 16 days. These signs were weakness, loss of appetite, dehydration, significant reduction in body weight, nose bleeding, and death. The pathological and biochemical changes were most severe in kidney tissue. The kidney lesions were bilateral and multifocal. At two days, degenerative and necrotic changes of the tubular and glomerular epithelium, thickening of glomerular membrane, vascular congestion, and edema were observed. At five days, proliferation of tubular epithelial and interstitial cells was observed. At 12 days, the cellular proliferation in both cortex and medulla was significantly greater. Fibrosis was observed at glomerular tuft, preglomeruli, pretubules, and interstitium (cortex and medulla). At 25 days, the collagen deposition reached the highest level in all regions, cellular proliferation decreased, and thickening of the arteriolar wall became prominent. The renal lesions were coupled with changes in the levels of protein, RNA, DNA, and hydroxyproline. At 40 days, the kidney showed signs of recovery. Blood urea nitrogen levels were significantly elevated at 2-25 days post-treatment. Stained tissue sections from liver, lung, heart, spleen, thymus, lymph nodes, testes, and adrenal glands of the treated rats were examined microscopically and appeared normal. Biochemically, significant changes (p less than .05) in protein, RNA, DNA, and hydroxyproline were also observed in these organs. At lower dosage (0.6 mg V/kg per day for 16 days), similar but less severe pathological and biochemical changes in kidneys and other organs were observed. At 0.3 mg V/kg per day for 16 days, the changes in the tissues were detected only at the biochemical level. These results indicate that the toxic effects of vanadium are cumulative and that vanadium-produced fibrosis in tissues is dose-dependent.

Mammary tumors in a colony of beagle dogs.

In a lifetime study, female beagle dogs in a closed colony were administered 226radium and 90strontium. An unirradiated control group was included in the study. A total of 223 of 356 dogs at risk developed 1,112 mammary proliferative growths (hyperplastic nodules and neoplasms). There was no correlation between occurrence and types of lesions in radiation and control groups. The age range for first occurrence of lesions was 10.4 to 13.9 years; hyperplastic nodule and benign mixed tumor occurred 1 to 2 years earlier than other lesions. A multiplicity of growths of similar or different morphological type were common throughout the lifetime of the dog. The female beagles, collectively, developed 244 hyperplastic nodules, 78 adenomas, 694 benign mixed tumors, 78 carcinomas, 14 malignant mixed tumors, and four myoepitheliomas. Proliferations occurred with increasing frequency from the cranial to caudal mammary glands. Metastasis was found in 77% of the dogs with carcinoma. The median time from diagnosis to metastasis was 10 months, but was shorter in dogs with infiltrative carcinoma.

Spontaneous cell-mediated cytolysis by peripheral blood cells obtained from whole-body chronically irradiated beagle dogs.

The level of natural killer (NK) activity of continuously gamma-irradiated (whole body) beagle dogs and their nonirradiated controls was studied. For analytical purposes, irradiated dogs were segregated into groups according to their clinical status: clinically normal, hypocellular, or with acute non-lymphocytic leukemia. Since unirradiated control animals exhibited a wide range of NK responses, the data from each irradiated animal were compared to its own age-matched or litter-matched unirradiated control. Of the eight clinically normal irradiated dogs (median = 146% activity of control) only one animal had a NK activity lower than that of its control. The hypocellular group (n = 5, median = 21.8% of control) and the leukemic group (n = 4, median = 52.5% of control) each contained one responder with higher activity than its control. The difference between the percentage of control of the clinically normal and clinically abnormal dogs was found to be significant (P less than 0.05). There is a negative correlation between the NK results obtained and the total accumulated dose of radiation at the time of sampling (correlation coefficient = -0.739, P less than 0.01), suggesting a radiation effect upon natural killer activity, which is evidence by enhancement at lower doses and depression at higher doses of irradiation.

Effect of thymopoietin and interleukin 2 on depressed mitogenic responsiveness and colony formation of lymphocytes from patients with preleukemia.

Previously, cloning efficiencies and mitogenic responsiveness of lymphocytes from patients with preleukemic disorders were shown to be significantly depressed. Whole blood T lymphocyte colony formation and 3H-TdR incorporation were used to assess the effects of interleukin-2 (IL-2) and thymopoietin (TP-5) on the proliferation of lymphocytes from patients with preleukemia. There were no statistically significant differences (p greater than 0.05) between any of the test groups stimulated with mitogen alone when compared to groups stimulated with mitogen plus TP-5 and/or IL-2 in either assay system for either patient or control groups. Nevertheless, TP-5 and IL-2 markedly increased the cloning efficiency and mitogenic responsiveness of lymphocytes from many of the patients studied, but in no case restored the proliferation response to the level of mitogen stimulated control lymphocytes. These findings suggest that other soluble mediators/factors may be needed to fully compensate for deficient mitogenic responsiveness and colony formation of lymphocytes from patients with preleukemic disorders which may be multifactoria in origin. Of importance, enhancement of lymphocyte responsiveness to PHA/Con-A with TP-5 and IL-2 suggests the presence of maturational/functional defects in lymphocytes from some of these patients which may be compensated for in part by addition of TP-5 and IL-2.

Kinetics of lymphohematopoietic progenitor cell populations in chronically irradiated RF/J mice.

Continuous protracted gamma irradiation (17.5 rad/22 h day for 28 days) resulted in significant life-shortening in RF/J mice due to lymphohematopoietic malignancies. The latency period of these neoplasms was decreased in irradiated RF/J versus unirradiated RF/J mice. No effect on leukemia incidence was observed in either irradiated or unirradiated CAF1 mice that served as control animals representing a strain with normal baseline lymphohematopoiesis. Lymphohematopoietic progenitor cell populations (CFU-GM and CFU-BL) were quantitated in unirradiated and chronically irradiated mice of both strains. The most remarkable differences in these parameters were seen with respect to CFU-BL. Unirradiated and irradiated RF/J mice produced over three times as many CFU-BL as CAF1 mice. Tremendously expanded lymphoid progenitor cell compartments in the RF/J mice may reflect the presence of numerically increased sensitive targets subject to radiation-induced damage and transformation. During a 12-week recovery period, CFU-BL and CFU-GM in the RF/J mice exhibited enhanced regenerative capabilities and overcompensatory responses that surpassed homeostatic baseline levels. Despite strain and strain X dose differences in CFU-BL and CFU-GM, no significant strain X dose relationships were seen in circulating leukocyte counts. This heightened proliferative activity and temporary overstimulation of radiation-damaged lymphohematopoietic tissues may allow sufficient promotional effect for leukemogenesis.

Increased in vitro radioresistance of bone marrow fibroblastic progenitors (CFU-F) from patients with acute non-lymphocytic leukemia.

The proliferative potential following in vitro irradiation of bone marrow fibroblastic progenitors (CFU-F) derived from four patients with acute nonlymphocytic leukemia (ANLL) and seven nonleukemic subjects was compared. The CFU-F from the ANLL patients were significantly more radioresistant than the CFU-F from the nonleukemic subjects. The increased radioresistance in ANLL patients was evident in both the mean slope of the survival curve (control = -0.385, ANLL = -0.256) and in the Do values (control = 2.68 Gy, ANLL = 4.61 Gy). Thus CFU-F derived from ANLL patients differ from those derived from nonleukemics in both radioresistance and in granulopoietic effects as suggested from previous studies.

In vitro radiation response studies on bone marrow fibroblasts (CFU-F) obtained from normal and chronically irradiated dogs.

The radiation resistance of bone marrow fibroblasts as measured by their proliferative potential was evaluated in chronically irradiated dogs. Bone marrows were obtained from eight dogs that had been chronically irradiated beginning at 21 days of gestation or after birth and eight age-matched controls. Of these irradiated dogs, four were either preleukemic or exhibited frank acute nonlymphocytic leukemia. The other four were clinically normal but demonstrated abnormalities in their marrow that could be attributed to radiation effects and/or other pathologic changes. Fibroblasts from six of the irradiated dogs were significantly more radioresistant than those of their controls. Five of these six dogs subsequently succumbed to hematopathologic disease, while the two irradiated dogs with normal fibroblasts remained clinically normal, suggesting that this observed radioresistance may be linked to the disease process.

Esophageal/gastric leiomyoma in the laboratory Beagle.

In a colony of 306 laboratory Beagles, gastric leiomyoma was diagnosed at necropsy (n = 69) and by biopsy (n = 1). The prevalence in males and females was nearly identical (23% and 22%, respectively). The neoplasm was strongly age related (P less than 0.001) and was seen in 82.4% of dogs aged 17 to 18 years. In contrast to malignant gastric tumors that generally are found in the distal two thirds of the stomach, the leiomyomas were found at the esophageal/gastric junction in 94% of cases. Clinical signs could not be specifically attributed to these masses, though their recognition was important in the differential diagnosis from other more serious gastric neoplasms.

Sample size for estimating the quantiles of endothelial cell-area distribution.

The estimation of corneal endothelium mean cell area (and, hence, mean cell density) is an important problem in clinical ophthalmology. Mitotic division of these cells is not known to occur, and cell deaths are followed by the enlargement of adjacent cells. As a consequence, cell-area distributions change drastically as functions of age and disease. Changes in cell-area distributions, in particular multimodality and skewness due to aging, are observed, and give rise to some difficult sampling problems. In this paper, sample quantiles are investigated as an alternative to the use of the sample mean. Asymptotic approximations are provided for the sample sizes required to estimate population quantiles with a desired precision. Asymptotic sample sizes are then compared with those obtained from tolerance limits. Empirical sample quantiles that can be used as benchmarks to compare corneas of normal individuals against corneas with unknown cell-area distributions are also presented. Aspects that merit further investigation are noted.

Effects of X irradiation on the growth of normal and hyperplastic mouse mammary gland transplants.

To avoid the problems associated with whole-body radiation, pieces of X-irradiated normal or hyperplastic mammary tissue were transplanted to the host gland-free fat pad of nonexposed mice. The percentage of the fat pad filled by growth of the transplants at 4, 8, and 12 weeks after transplantation was measured. Growth of lobule transplants was moderately inhibited by 4 Gy. While some of the lobules survived 12 Gy, their growth was severely inhibited. The hyperplastic outgrowth lines were variable but more resistant than lobules to growth retardation. Line Z5D was more susceptible than D1, and Z5C1 was least susceptible, with 88% growing well after 12 Gy. In order to distinguish between transient and permanent growth retardation, tissue was taken from the irradiated and control transplants and retransplanted to new hosts without further radiation. The second generation of X-ray-exposed tissue filled more of the fat pad than the first-generation transplants, but significantly less than the nonexposed controls. The experiments described provide a means of demonstrating X-ray-induced changes in the mammary gland from growth inhibition to carcinogenesis.

Studies of T-lymphocytes in preleukemic disorders and acute nonlymphocytic leukemia: in vitro radiosensitivity, mitogenic responsiveness, colony formation, and enumeration of lymphocytic subpopulations.

Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and lymphocyte colony formation (CFU-L) from whole blood were measured following in vitro x-irradiation. Lymphocytes from patients with myelodysplastic disorders, acute nonlymphocytic leukemia, and patients at increased risk for leukemia because of their primary disease and/or cytotoxic therapy were found to be significantly more sensitive to in vitro x-irradiation than lymphocytes from clinically normal individuals. Cloning efficiencies and mitogenic responsiveness of patient lymphocytes were significantly depressed as compared to normal values. Using monoclonal antibodies to specific surface markers, quantitative abnormalities in lymphocytic subpopulations from myelodysplastic patients also were observed. These findings are suggestive of a defect at the T-cell level that may directly or indirectly affect hematopoiesis.

Acute toxicity of ammonium metavanadate in mice.

Acute toxicity of ammonium metavanadate solutions in normal saline (pH 6.7) or 0.1 M Tris-HCl-NaCl buffers (pH 7.2 or pH 7.8) was studied in BALB/c mice at 20 mg V/kg. Animals receiving these solutions subcutaneously started to show severe clinical signs 10-15 min postinjection and high mortality rates (45-73%) during the first 3 d. Animals dying because of vanadium toxicity did so only within the first 3 d after injection. NH4VO3-treated animals showed a tendency to increase their liver and spleen weights as compared to those receiving control solutions. Severe necrosis in lymphoid tissues (thymus, spleen, lymph nodes, and Peyer's patch), pulmonary hemorrhage, and renal acute tubular necrosis were commonly demonstrated in vanadium-treated animals. Toxicity of NH4VO3 solution in 0.1 M Tris-HCl-NaCl buffer (pH 7.8) was greatly reduced upon acidification with HCl to pH 6.1 or following boiling for 15 min (final pH of 7.7). Acidification of the solution reduced the mortality rate to 20 from 68%; however, the clinical signs were still severe. Boiling of the solution reduced the mortality rate to zero and moderated the severity of the clinical signs.

Radiation-induced inhibition of human lymphocyte blastogenesis: the effect of superoxide dismutase and catalase.

Mitogen-induced lymphocyte blastogenesis was measured following X-irradiation (0-4 Gy) in the presence or absence of superoxide dismutase (SOD), under aerobic and anaerobic conditions. There were no significant differences between radiation survival curves under these different conditions, nor did SOD have any radioprotective effect. This demonstrates the lack of oxygen dependence of radiation-induced inhibition of lymphocyte blastogenesis. Following X-irradiation at 2 Gy, neither SOD nor catalase, alone or together, added before or after irradiation, were radioprotective. In comparison to controls, both enzymes depressed lymphocyte proliferation when added at levels as low as 25 microgram catalase or 100 microgram SOD/ml media. When SOD and catalase were added together, the greatest depression of blastogenesis was obtained with increasing levels of SOD relative to increasing levels of catalase, indicating that SOD was largely responsible for this depression. The suppressive effect of administration of SOD (p less than 0.05), catalase (p less than 0.001) and SOD + catalase (p less than 0.001) on lymphocyte division was significantly greater when given prior to X-irradiation. The lack of an oxygen effect and the inability of SOD and catalase to protect human lymphocytes from X-irradiation suggest that 2- and /or H2O2 are not involved in radiation-induced inhibition of lymphocyte blastogenesis.