David V. Tolle

Hematopoietic responses under protracted exposures to low daily dose gamma irradiation

In attempting to evaluate the possible health consequences of chronic ionizing radiation exposure during extended space travel (e.g., Mars Mission), ground-based experimental studies of the clinical and pathological responses of canines under low daily doses of 60Co gamma irradiation (0.3-26.3 cGy d-1) have been examined. Specific reference was given to responses of the blood forming system. Results suggest that the daily dose rate of 7.5 cGy d-1 represents a threshold below which the hematopoietic system can retain either partial or full trilineal cell-producing capacity (erythropoiesis, myelopoiesis, and megakaryopoiesis) for extended periods of exposure (>1 yr). Trilineal capacity was fully retained for several years of exposure at the lowest dose-rate tested (0.3 cGy d-1) but was completely lost within several hundred days at the highest dose-rate (26.3 cGy d-1). Retention of hematopoietic capacity under chronic exposure has been demonstrated to be mediated by hematopoietic progenitors with acquired radioresistance and repair functions, altered cytogenetics, and cell-cycle characteristics. Radiological, biological, and temporal parameters responsible for these vital acquisitions by hematopoietic progenitors have been partially characterized. These parameters, along with threshold responses, are described and discussed in relation to potential health risks of the space traveler under chronic stress of low-dose irradiation.

Circulating Micromegakaryocytes Preceding Leukemia in Three Dogs Exposed to 2.5 R/day Gamma Radiation

The presence of micromegakaryocytes in the blood and bone marrow of humans during the preleukemic phase of acute and chronic myelogenous leukemia and myelomonocytic leukemia is well documented [I, 4, 5, 7, 8, 10, 15), and it has been suggested that it is the single most typical abnormality suggesting a subsequent course towards overt leukemia (6). Three adult purebred beagles that died with myelogenous leukemia during continuous whole-body exposure to 2.5 roentgens/22-hour day of 6OCO gamma irradiation had micromegakaryocytes and megakaryoblasts in the peripheral blood three to ten weeks before leukemic myeloblasts were observed in buffy coat preparations. The cells of interest were 6 to 30 JLm in diameter, mono-, or binucleated, with round or oval nuclei, indistinct nucleoli, and they had a high nuclear/cytoplasmic ratio (fig. I). Many cells had cytoplasmic blebs (fig. 2). Cytochemical characterization revealed strong positive staining for both acetylcholinesterase, and a-naphthyl acetate esterase, as well as abundant glycogen with the periodic acid-Schiff stain. The cells were negative with Sudan black, and no detectable myeloperoxidase or naphthol AS-D chloroacetate esterase activity was seen. Other hematologic abnormalities observed during this preleukemic period included progressive refractory anemia, occasional nucleated red blood cells, anisopoikilocytosis, monocytosis, giant platelets, and a left shift to metamyelocytes. Over the same period, bone marrow preparations from iliac crest aspirates and rib biopsies revealed granulocytic hyperplasia with a moderate left shift but without an excess of myeloblasts. There was also erythroid depletion, and increased numbers of mono-, bi-, and multinucleated megakaryocytes (fig. 3). Ultrastructural characterization clearly indicated the cells to be micromegakaryocytes and megakaryoblasts. Even in the most immature cells, the cytoplasm contained both dense serotonin granules and the characteristic a (bulls-eye) granules, indicating their megakaryocytic lineage (fig. 4). Although the majority of micromegakaryocytes showed dysgenesis of the demarcation membrane system, the cytoplasm did contain smooth, membranous vesicular sacs of a rudimentary demarcation membrane system (fig. 4) and in a few cells there were flattened, laminar cisternae of the demarcation membrane system with peripheral formation of dystrophic (i.e., agranular, giant) platelets. Terminally, the dogs were severely anemic (0.94, 1.10, and 1.70 X 10 red blood cells/ul), and thrombocytopenic (5.0, 7.5, and 90.0 X 10 Total leukocyte counts were 11.5, 14.8, and 57.1 X 10/ JLI, respectively. The terminal peripheral blood differential leukocyte count showed a marked left shift (17%myeloblasts) in only one dog. However, bone marrow imprints obtained at necropsy from sternum, rib, and femur showed, in all dogs, a granulocytic hyperplasia with maturation defects and myeloblast counts of 21.0, 11.0, and 35.5% of the total granulocytic series. In all dogs there was a paucity of erythroid elements and in only one dog were there a few remaining megakaryocytes.

Acute Monocytic Leukemia in an Irradiated Beagle

A purebred female Beagle dog that had received 2,000 R of protracted whole-body γ-irradiation from 60Co when 14 months old had hematologic changes consistent with a myeloproliferative disorder 3 years after the termination of radiation exposure. Peripheral blood and bone marrow findings during the 7-month period before death showed progressive anemia with increased numbers of platelets; immature granulocytes, monocytes and promonocytes. A period of partial remission occurred during which time the peripheral blood was aleukemic, although there was marked thrombocytosis and abnormal erythropoiesis which was evidenced by bizarre circulating nucleated red cells, anisocytosis, poikilocytosis and Howell-Jolly bodies. The dog had a terminal crisis with marked leukocytosis, most cells in the peripheral blood being bizarre monocytes and promonocytes. Tissues obtained at necropsy showed diffuse as well as focal infiltration of the spleen, liver, lymph nodes, heart, kidney and gastrointestinal wall with immature neoplastic cells resembling monocytes and monocytic precursors. The monocytic differentiation of the invasive cell population was confirmed by morphological, cytochemical, histological, ultrastructural and in vitro cell culture studies.

Chronic radiation leukemogenesis: Postnatal hematopathologic effects resulting from in-utero irradiation☆

Previous studies have shown that continuous whole-body exposure to low daily doses of gamma radiation is highly leukemogenic for beagles initially exposed during either young adulthood or fetal development. In contrast, terminated radiation-exposure regimens (continuous exposure terminated after accumulation of preset total radiation doses) markedly reduce leukemogenic potential. In this study, we examined leukemic incidences and postnatal hematopoietic function in three groups of dogs; continuously irradiated (7.5 cGy/day) during both fetal life and after birth, continuously irradiated during fetal life only, and nonirradiated. Results were compared to results from studies with similarly irradiated and nonirradiated groups of young adult dogs initially tested at 400 days of age. Hematopoietic function was assessed in terms of both circulating blood levels of red cells, platelets, granulocytes, and monocytes, and marrow concentrations and radiosensitivities of hematopoietic progenitors. Results indicated that under continuous fetal/postnatal irradiation, i.e. the high leukemogenic exposure regimen, a marked, progressive suppression in hematopoietic function occurred following birth. This suppression continued to 100-150 days of age and was followed by partial hematopoietic recovery that was associated with an acquired radioresistance by hematopoietic progenitors. In contrast, neonates that had been continuously irradiated during fetal life, but not postnatally, i.e. the low leukemogenic regimen, exhibited a similar initial suppression of hematopoietic function followed by partial recovery. However, no temporally linked acquisition of radioresistance by hematopoietic progenitors was demonstrated. These results support the hypothesis, developed from earlier studies with adult dogs, that the processes of acquired radioresistance and recovery in numbers of transformable hematopoietic progenitors are causally linked to early stages of the leukemogenic process under continuous ionizing irradiation.

Survival Patterns and Hemopathological Responses of Dogs Under Continuous Gamma Irradiation

Survival curves were constructed and analyzed relative to contributing hematopathological responses for groups of beagles exposed continuously for duration of life to low daily doses of whole body 60Co gamma irradiation (27.3 rads/day to 4 rads/day). The survival curves versus time were progressively displaced toward longer survival as rates of exposure were reduced from the relatively high dose rate of 27.3 rads/day to the low dose rate of 4.0 rads/day. Average survival times increased from 57 days at 27.3 rads/day to 1830 days at 4.0 rads/day, representing fractional increased life-spans from 1.5% to 50.8%, respectively. Survival curves versus total dose were markedly displaced along the cumulative radiation dose axis at the extreme dose rates (i.e., 27.3 and 4.0 rads/day), but not at the intermediate dose rates (i.e., 13.4 and 7.9 rads/day) in which the upper linear portions of the survival curves are superimposed. From these dose-dependent survival curves, LD 50 values for whole body gamma irradiation, delivered chronically at 27.3, 13.4, 7.9, and 4.0 rads per day were estimated to be 1442, 2124, 2039, and 7161 rads, respectively. Both time- and dose-dependent survival curves for the intermediate dose rates, in contrast to the extreme dose rates, exhibited pronounced transitions in the lethality rate below the 50% survival level. These lethality rate transitions occurred at ~ 2500 rads of accumulated dose and were attributed to a shift in the spectrum of developing hematopathologies: namely, from a predominance of the acutely ablative radiation-induced lymphohematopoietic syndromes (i.e., septicemias and aplastic anemias) to a predominance of the late arising hematopoietic neoplasias (myelogenous leukemia and related myeloproliferative disorders). Based on the concept that these lethality rate transitions and shifts in the spectrum of developing pathologies reflect distinct subgroups of dogs of varying radiosensitivities and pathological tendencies, estimates of LD values for subgroups with specific pathologies are given (e.g., 1850 rads for the subgroup with aplasia; 5500 rads for the subgroup with myeloproliferative diseases). Sequential analyses of blood and marrow responses indicated that this shift in hematopathologic spectrum (and in turn the lethality rate transition) is due to an early reparative hematological event that is prerequisite to prolonged survival and, in turn, to the expression of late arising neoplasias. The cellular basis of this repair appears to be mediated, in part, by an acquisition of radioresistance by early hematopoietic progenitors.

Hemorrhagic Diathesis in Laboratory Rodents

Summary A disease characterized by spontaneous hemorrhage and death occurred in newly established breeding colonies of cesarean-derived, specific-pathogen-free, Sprague-Dawley rats and CF no. 1 mice. Male mice and rats were more commonly affected than females, and pregnant females were more commonly affected than nonpregnant females. In addition to a wide variety of hemorrhages, there was a high frequency of myocardial lesions in the decedent male mice. The clotting mechanism was studied and the defect was shown to be related to factors II and VII and possibly factor X. Menadione was effective in treating the disease but did not completely prevent it. The possible predisposing and etiologic factors, including the alteration of intestinal bacterial flora, are discussed, and the need to fully characterize animals used in experiments is stressed.

Blood responses under chronic low daily dose gamma irradiation: I. Differential preclinical responses of irradiated male dogs in progression to either aplastic anemia or myeloproliferative disease

Male beagles chronically exposed to low daily doses of 60Co gamma rays (7.5 cGy/22h/day) show one of three hematopoietic patterns, which reflect three different distinctly responding subgroups: (1) low radioresistance with progressing aplastic anemia and shortened survival (-S-AA subgroup); (2) high radioresistance with a complex of progressing myeloproliferative disorders (+R-MPD group); or (3) high radioresistance with other nonMPD syndromes (+R-nonMPD group). Blood cell levels (granulocytes, monocytes, erythrocytes, lymphocytes, and platelets) were assessed and fitted to a flexible polynomial spline model, thus defining the (a) initial suppressive and (b) subsequent recovery phases for the subgroups. Results showed that relative to the overall magnitude of blood cell loss as well as to the maximum rate of suppression during the initial phase, the subgroups were generally ranked -S-AA much greater than +R-MPD greater than +R-nonMPD. Relative to the overall strength of the recovery response, the subgroups were generally ranked +R-MPD greater than +R-nonMPD much much greater than -S-AA. In terms of overall maintenance levels of circulating blood cells during the recovery phase, however, the +R-nonMPD subgroup consistently exhibited stronger responses than the +R-MPD subgroup. These results tend to support our contention that selected subgroups of dogs have strong propensities to specific hematopathologies (i.e. aplastic anemia and myeloid leukemia) under chronic irradiation and that these pathology-prone animals exhibit a series of marked differential hematopoietic responses during early preclinical phases, which serve effectively to prognosticate subsequent pathological progression.

Irradiation-Induced Canine Leukemia: A Proposed New Model. Incidence and Hematopathology

A leukemia model in a large animal, analogous to human myelogenous leukemia, would be invaluable for not only leukemogenic studies, but also in the design of chemotherapeutic trials and supportive therapy. The incidence of spontaneous myeloid leukemia in most domestic animals, however, is quite low (15,17,26), and this disease rarely occurs in the dog (10).