The induction of liver tumors by 239Pu citrate or 239PuO2 particles in the Chinese hamster

The influence of radiation dose distribution on the frequency of 239Pu-induced liver tumors was evaluated in the Chinese hamster. Different concentrations of 239Pu citrate 239PuO2 particles of known sizes were injected intravenously via the jugular vein. About 60% of the injected 239Pu citrate was deposited in the liver and 40% in the bone. The 239Pu citrate was rather uniformly distributed throughout the liver parenchyma. Injected plutonium oxide particles were taken up by the reticuloendothelial system with 90% of the body burden deposited in the liver. The 239PuO2 particles were localized in the Kupffer cells and produced nonuniform dose distributions that were dependent on particle size. There was an activity- and dose-dependent increase in the incidence of total liver parenchymal cell tumors following injection with either plutonium particles or citrate. For animals that received 14.0-, 2.7-, 0.3-, and 0.04-Gy dose to liver from 239Pu citrate the cumulative tumor incidence was 39, 32, 5, and 0%, respectively. Animals that were injected with the 0.24 micron 239PuO2 particles had doses of 42.0, 7.2, and 0.8 Gy to the liver and tumor incidences of 34, 26, and 5%, respectively. Plutonium citrate also produced hemangiosarcomas of the liver and tumors in bone and bone marrow. The latent period for liver tumor appearance in animals exposed to 239Pu citrate or 239PuO2 particles increased as the injected activity decreased. For animals injected with a similar total activity (7.4 Bq/g), the lifetime cumulative liver tumor incidence was similar for animals exposed to either 239Pu citrate (32%) or 239PuO2 (26%). There was little effect of particle size on liver tumor incidence. These data indicate that, in Chinese hamster liver, local radiation dose distribution is less important in altering tumor incidence than injected activity or average dose. However, the more uniform irradiation from 239Pu citrate administration was more effective in cancer production than the nonuniform irradiation from 239PuO2 particles.     

Long-term effects of low-level 239Pu contamination on murine bone-marrow stem cells and their progeny

The effects of long-term internal contamination with 13.3 kBq kg-1 239Pu injected intravenously were studied in 10-week-old ICR (SPF) female mice. Radiosensitivity of spleen colony-forming units (CFU-S) and 125IUdR incorporating into proliferating cells of vertebral bone marrow and spleens were determined in plutonium-treated and control animals one year after nuclide injection. The CFU-S in 239Pu-treated mice were more sensitive to X-rays (D0 = 0.52 +/- 0.01 Gy) than in controls (D0 = 0.84 +/- 0.02 Gy). 125IUdR incorporation into bone marrow and spleen cells was reduced after plutonium contamination. At one year following plutonium injection, the occurrence of chromosome aberrations was evaluated in metaphase figures of femoral bone marrow cells. The frequency of aberrations increased early after plutonium treatment, at later intervals it tended to decrease but not below the control level. While the relative numbers of vertebral marrow CFU-S decreased significantly, but only to 86 per cent of normal, cellularity of vertebral bone marrow, peripheral blood counts and survival of 239Pu-treated mice did not differ from the control data.     

Quantitative microscopic studies of the distribution and retention of 239Pu in the ilium of the female CBA mouse.

Three month old female CBA mice were injected with 50 nCi kg-1 body mass of minimally polymeric 239Pu-citrate and killed at 24 hours, 10 days, 1 month and 3 months after injection. The distribution of 239Pu in the ilia of these mice was analysed using neutron-induced autoradiography of bone sections together with computer-based methods of data reduction. Results of these investigations demonstrate that while 239Pu is initially localized on bone surfaces, by 3 months after injection it is fairly uniformly distributed throughout mineral bone and its included marrow.     

The relationship between 59Fe uptake in marrow and 239Pu deposition in bone.

The marrow in the left femur of each of 17 mice was destroyed by X-irradiation and 59Fe and 239Pu uptake into both femurs was measured 1, 3 and 7 days later. Uptake of 59Fe into marrow was depressed in the left femur 1 and 3 days after irradiation but was enhanced in the right unirradiated femur 3 days after the left femur was irradiated. There was no corresponding depression of 239Pu uptake into the left irradiated femur or enhancement into the right unirradiated femur. These results do not support the view that a functioning erythropoietic marrow is necessary for 239Pu to be deposited in bone.     

Radioprotection of mice with interleukin-1: relationship to the number of spleen colony-forming units

Compared to saline-injected mice 9 days after 6.5 Gy irradiation, there were twofold more Day 8 spleen colony-forming units (CFU-S) per femur and per spleen from B6D2F1 mice administered a radioprotective dose of human recombinant interleukin-1-alpha (rIL-1) 20 h prior to their irradiation. Studies in the present report compared the numbers of CFU-S in nonirradiated mice 20 h after saline or rIL-1 injection. Prior to irradiation, the number of Day 8 CFU-S was not significantly different in the bone marrow or spleens from saline-injected mice and rIL-1-injected mice. Also, in the bone marrow, the number of Day 12 CFU-S was similar for both groups of mice. Similar seeding efficiencies for CFU-S and percentage of CFU-S in S phase of the cell cycle provided further evidence that rIL-1 injection did not increase the number of CFU-S prior to irradiation. In a marrow repopulation assay, cellularity as well as the number of erythroid colony-forming units, erythroid burst-forming units, and granulocyte-macrophage colony-forming cells per femur of lethally irradiated mice were not increased in recipient mice of donor cells from rIL-1-injected mice. These results demonstrated that a twofold increase in the number of CFU-S at the time of irradiation was not necessary for the earlier recovery of CFU-S observed in mice irradiated with sublethal doses of radiation 20 h after rIL-1 injection.     

Self-renewal capacity of murine hemopoietic stem cells under internal contamination with239Pu and241Am

Summary The self-renewal capacity of murine pluripotent hemopoietic stem cells (CFU-S) of vertebral bone marrow was studied under conditions of short-term and long-term internal contamination with²³⁹Pu or²⁴¹Am in female mice. Measurement of the CFU-S self-renewal capacity was carried out using double transplantation assay. To evaluate the production of differentiated progeny of stem cells average erythroblast numbers/visible spleen colony and⁵⁹Fe-uptake/colony were computed. The marrow cellularity/vertebra and the number of CFU-S/vertebra were decreased and affected more by²³⁹Pu than by²⁴¹Am. The production of erythroblasts per a single CFU-S and the⁵⁹Fe-uptake/colony were reduced, similarly the numbers of secondary spleen colonies and of secondary CFU-S in primary colonies. The above changes resulting from impaired functions of surviving CFU-S were more serious with²⁴¹Am than with²³⁹Pu. The biological effects of plutonium and americium appeared independent of the phase of contamination.     

Radiosensitivity and proliferative activity of vertebral CFU-S surviving in mice injected with oncogenic activities of 239Pu or 241Am

Survival, radiosensitivity and capability to produce differentiated progeny were followed in CFU-S from lumbar vertebrae of mice injected with 198.6 kBq 239Pu/kg or 208.6 kBq 241Am/kg. The CFU-S assay and 59Fe uptake into spleen colonies were used. The number of CFU-S from treated mice was significantly lower than in controls. Higher radiosensitivity of CFU-S from 239Pu- or 241Am-treated mice was demonstrated using additional exposure to 0.5 Gy X-rays 1, 24, 48, 72 hrs after cell transplantation and expressed more precisely by survival curves obtained 1 hr after the marrow cell injection. The effect of 239Pu on CFU-S was characterized by Do 0.58 Gy (n = 0.91) and that of 241Am by Do 0.64 Gy (n = 0.91); corresponding control values were Do 0.89 Gy, n = 1.11. Lower iron utilization due not only to the decreased CFU-S numbers, but also to the defective production of erythroid cells per one CFU-S was found. Complexity of radiation effect on hemopoietic stem cells was demonstrated by the present study.     

Failure of ovalbumin associated with allogeneic spleen ceels to stimulate proliferation of lymph node cells of immune mice.

Ovalbumin-pulsed spleen cells were found to stimulate thymidine uptake of lymph node cells of syngeneic mice immunized with ovalbumin in complete Freund's adjuvant after treatment of spleen cells with Mitomycin C but not after heating the spleen cells at 56degrees for 30 min. Ovalbumin-pulsed spleen cells of allogeneic mice failed to stimulate the immune lymph node cells more than unpulsed cells, although a net increase in the thymidine uptake above the allogeneic stimulation was observed when free ovalbumin was added to the mixed culture. To eliminate the high background of the mixed lymphocyte reaction, F1 mice were made chimeric with bone marrow of one of the parental strains. Using lymph node cells of the immunized chimeras, the stimulation by pulsed spleen cells was much greater when antigen was presented on cells of the parental strain used for bone marrow injection than when presented on cells of the other parental strain.     

Characteristics of the reactions to N-alloantigens of lymphoid cells from hydrocortisone-stimulated and adrenalectomized mice]

The capacity of the spleen, bone marrow and thymus cells from CBA mice (intact, adrenalectomized, and those treated with single or repeated hydrocortisone injections) to induce the lymph node type of "graft-versus-host" reaction (GVHR) in (CBA X C57BL) F1 hybrid recipients was evaluated. Two days after 2.5 mg hydrocortisone injection the capacity of the spleen and bone marrow cells to induce GVHR increased while that of the thymus cells remained unchanged. Seven and particularly 15 days after hydrocortisone injection the spleen cells became less active. Two days following repeated daily hormone injections in a dose of 0.25 mg within 18 days the thymocyte activity in GVHR increased, while that of the spleen and bone marrow cells did not change.     

Radioprotection of mice with interleukin-1: relationship to the number of erythroid and granulocyte-macrophage colony-forming cells

This report presents the results of an investigation of changes in the number of erythroid and granulocyte-macrophage colony-forming cells (GM-CFC) that had occurred in tissues of normal B6D2F1 mice 20 h after administration of a radioprotective dose (150 ng) of human recombinant interleukin-1 (rIL-1). Neutrophilia in the peripheral blood and changes in the tissue distribution of GM-CFC demonstrated that cells were mobilized from the bone marrow in response to rIL-1 injection. For example, 20 h after rIL-1 injection marrow GM-CFC numbers were 80% of the numbers in bone marrow from saline-injected mice. Associated with this decrease there was a twofold increase in the number of peripheral blood and splenic GM-CFC. Also, as determined by hydroxyurea injection, there was an increase in the number of GM-CFC in S phase of the cell cycle in the spleen, but not in the bone marrow. Data in this report suggest that when compared to the spleen, stimulation of granulopoiesis after rIL-1 injection is delayed in the bone marrow. Also, the earlier recovery of GM-CFC in the bone marrow of irradiated mice is not dependent upon an increase in the number of GM-CFC at the time of irradiation.     

Effect of plutonium-239 on the mitotic activity of mouse bone marrow cells

Summary Mitotic index of the bone marrow cells was studied in femoral bone marrow of mice given 313 kBq²³⁹Pu kg^–1. The attention was turned to the femoral midshaft and the mitose concentration, intensified by Colcemid stathmokinetic effect, was evaluated in a sampling field from endosteal surface to the central venous canal, throughout 68 weeks. It has been found that the plutonium effect in the sampling band is rather uniform except the points in subendosteal zone early after plutonium injection, where the mitotic index was reduced in such a way that the mitotic gradient, observed in controls, was affected. The mitotic activity in femoral diaphysis of plutonium injected mice was mobilized approximately till the 30th week of contamination. Later it deteriorated progressively. The results are discussed and should not be regarded as representative for the entire bone marrow hemopoiesis.     

Effect over time of in-vivo administration of the polysaccharide arabinogalactan on immune and hemopoietic cell lineages in murine spleen and bone marrow.

Current evidence indicates an immunostimulating role for complex carbohydrates, i.e., polysaccharides, from several plant sources. In the present work, we determined the specific in vivo effects, with time of administration, of one such compound, a neutral arabinogalactan from larch not only on immune (lymphoid) cells, but also on natural killer (NK) lymphoid cells, as well as a variety of other hemopoietic cells in both the bone marrow and spleen of healthy, young adult mice. The latter were injected daily (i.p.) with arabinogalactan (500 microg in 0.1 ml pH 7.2 phosphate buffered saline-PBS) for 7 or 14 days. Additional, aged (1 1/2-2 yr) mice were similarly injected for 14 days only. Control mice were given the PBS vehicle in all cases, following the above injection regimen. Animals from all groups were sampled 24 h after the final injection and the immune and hemopoietic cell populations in the bone marow and spleen were assessed quantitatively. The results indicated that immediately following either 7 or 14 days of arabinogalactan administration to young, adult mice, lymphoid cells in the bone marrow were significantly decreased (p < 0.004; p < 0.001, respectively) relative to controls but remained unchanged at both time intervals in the spleen. NK cells, after 7 days of arabinogalactan exposure, were also decreased significantly in the bone marrow (p < 0.02), but unchanged in the spleen. After 14 days' exposure to the polysaccharide, NK cells in the bone marrow had returned to normal (control) levels, but were increased in the spleen (p < 0.004) to levels greater than 2-fold that of control. Among other hemopoietic cell lineages, none was influenced in the bone marrow or spleen by one-week administration of arabinogalactan; however, after two-week exposure, precursor myeloid cells and their mature (functional) progeny (granulocytes), were significantly reduced in the spleen (p < 0.043; p < 0.006, respectively), as were splenic monocytes (p < 0.001). These lineages in the bone marrow, however, remained steadfastly unaltered even after 14 days of continuous exposure to the agent. Of the vast cascade of cytokines induced in the presence of this polysaccharide, it appears that immunopoiesis- and hemopoiesis-inhibiting ones are most prevalent during at least the first two weeks of daily exposure.     

The origin of antibody-forming cells detected in the bone marrow after thymus-independent antigen-stimulation and abnormality in migration of B cells of X-linked immunodeficient CBA/N mice

The anti-TNP IgM plaque-forming cells (PFC) were generated in the spleen and bone marrow of non-immunodeficient normal mice after intraperitoneal administration of TNP-LPS. Irradiation of normal mice while shielding bone marrow completely abrogated the generation of bone marrow PFC, indicating that they are derived from extramedullary sites. The bone marrow PFC, response to TNP-LPS was low in X-linked immunodeficient CBA/N strain mice, while the spleen response was comparable to that seen in the normal mice. To further study the basis of the deficient bone marrow PFC response in CBA/N mice, spleen cells were adoptively transferred to irradiated syngeneic mice stimulated with TNP-LPS. While spleen cells from normal mice generated high numbers of PFC in recipient bone marrow and spleen, those from CBA/N strain mice could not generate bone marrow PFC. This result was obtained regardless of whether normal or CBA/N recipients were used. These results indicate that TNP-LPS administration normally results in the migration of B lymphocytes from the periphery into the bone marrow and that B cells from immunodeficient CBA/N strain mice bear an inherent defect in this migratory function. This migratory defect was shown to be X-linked, as are the other previously reported B cell defects in this inbred mouse strain. The possible relationship between this migratory defect and the maturational defects of B cell lineage as reported previously in CBA/N strain mice is discussed.     

Distributions of cell populations within alpha-particle range of plutonium deposits in the rat and beagle testis

Plutonium is not uniformly distributed in testicular tissues; thus some cell populations may receive larger or smaller radiation exposures than would be expected if the nuclide were uniformly distributed. The distributions of cell populations within alpha-particle range of Pu deposits in rat and beagle testes were determined. The data were collected from autoradiographs of testicular tissues containing 241Pu. A cell distribution factor (CDF) was determined for each cell population and is defined as the average number of each cell type within alpha-particle range of each observed Pu deposit relative to the number of each cell type that would be expected within alpha-particle range of each Pu deposit, if the deposits were distributed uniformly. In addition, the percentage of the spermatogonial stem cell population within alpha-particle range of Pu deposits was determined. In rats, the CDF for the spermatogonial stem cells is about 2.2. This value is similar to other enhancement and inhomogeneity factors reported for rodents in the literature. In beagles the CDFs to all cells in the seminiferous epithelium were less than the rats. In addition, the percentage of spermatogonial cells within alpha-particle range of Pu concentrations in the interstitial tissues was a factor of about 3 less in the dog than in the rat. The largest CDFs seen in both species were in the interstitial tissues, particularly for Leydig cells. Because the organization of testicular tissues in the beagle is quite different from rodents but more similar to human, the results from this study suggest that extrapolations from rodents to humans may tend to overestimate the potential for radiation exposure to spermatogonial stem cells as well as the fraction of the spermatogonial stem cell population at risk to exposure from internally deposited 239Pu.     

Antibody formation in mouse bone marrow. IX. Peripheral lymphoid organs are involved in the initiation of bone marrow antibody formation.

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during secondary-type responses, it becomes the major organ, containing IgM, IgG, and IgA PFC. In the present paper, the influence of splenectomy (Sx) upon the secondary bone marrow PFC response to SRBC was investigated. When previously primed mice were splenectomized just before the second intravenous (iv) injection of SRBC, the effect of Sx upon the height of the bone marrow PFC response was dependent on the booster dose. Sx just before a booster of 10⁶ SRBC iv almost completely prevented bone marrow PFC activity, whereas an iv booster dose of 4 × 10⁸ SRBC evoked a normal IgM, IgG, and IgA PFC response in Sx mice. Apparently low doses of iv administered antigen require the spleen in order to evoke antibody formation in the bone marrow. Experiments with parabiotic mice, consisting of Sx and sham-Sx mice, showed that this facilitating influence of the spleen upon bone marrow antibody formation occurs via the blood stream. In a subsequent study, it was investigated whether the spleen is required throughout the bone marrow PFC response or only during the few days of the initiation phase. Therefore, mice were splenectomized at different intervals after a booster injection of 10⁶ SRBC iv. It appeared that Sx 2 days after the booster injection could still prevent the normal bone marrow PFC activity, whereas Sx at Day 4 could no longer do so. Apparently, after an iv booster injection, the spleen is only required for initiation of the bone marrow PFC response and not for the maintenance of this PFC activity thereafter.     

KINETICS OF ERYTHROPOIETIN STIMULATED PROLIFERATION OF ERYTHROID PROGENITORS IN THE SPLEEN

The effect of RBC transfusion and erythropoietin (EPO) on the proliferation of immature erythrocyte progenitors was studied in the spleens of RBC transfused, lethally irradiated mice injected with bone marrow. Transfusion decreased expansion of the progenitors and slowed their proliferation: the mean cycle time as measured by per cent labelled mitosis (PLM) on the third day after injection of bone marrow was 10.7 hr in transfused as compared to 5.6 hr in non-transfused mice. One injection of five units of erythropoietin on day 2 decreased the mean cycle time to 7.3 hr in transfused mice and increased expansion of the progenitor cells. The effects of erythropoietin on cell proliferation were prompt: a significant increase of incorporation of ³H-TdR into DNA occurred within 2 hr of injection. Erythroblasts were absent from the spleens of transfused, irradiated bone marrow injected mice; however, erythroblasts appeared by 72 hr and 48 hr following EPO injection either 2 days or 5 days after transplantation respectively. Increased uptake of radioactive iron in spleen after erythropoietin injection preceded the appearance of erythroblasts by 2 and 1 days when erythropoietin was injected either 2 or 5 days after marrow transplantation respectively. The increase in cellular proliferation induced by erythropoietin in transfused irradiated mice injected with bone marrow equivalent to 0.35 femoral shaft was manifested as an increase of the total DNA content in the spleen by 119 μg (11.9 × 10⁶ cells) within 48 hr of injection. The cellular increment produced by EPO injection on day 5 to mice given 0.05 femoral shaft consisted mainly of undifferentiated mononuclear cells, most of which were labelled, with erythroblasts comprising only one quarter of the increment. Erythropoietin inactivated by mild acid hydrolysis failed to increase cellular proliferation.     

The development and interlobar distribution of plutonium-induced pulmonary fibrosis in mice

Six-week-old mice were exposed by inhalation to an aerosol of 239PuO2 (activity median aerodynamic diameter 2.2 microns) to establish mean alveolar depositions at 2 days after exposure of 4, 40, and 930 Bq of 239Pu. Animals were killed serially after 3, 6, 12, and 18 months at which times the development of the pulmonary fibrotic lesion was assessed by both biochemical and histopathological techniques. Individual measurements of both fresh and dry weights, protein, DNA, and hydroxyproline were made on whole lung and also on each of the five constituent lobes. Early and sustained increases in lung mass, lung protein, and total lung collagen were found, together with a depression of the total cellularity of the lung at 6 and 12 months after exposure. Although at later times compensatory hypertrophy of less affected areas distorted the relationship, systematic trends in the severity of responses between lobes were found. These trends were related to the initial lobar concentrations of 239Pu.     

Immune mechanisms in leukemia: protective capacity of the major lymphoid cell compartments.

The capacity of immune spleen, lymph node, peritoneal, bone marrow, and thymic cells to protect C58/wm mice from syngeneic transplanted line Ib leukemia was quantified. Cells harvested 14 to 15 days after primary immunization were used for adoptive protection tests. Regression curves were computer analyzed and log10, PD50 values compared. For immune spleen, lymph node, peritoneal, bone marrow, and thymic cells the PD50 values were 4.53, 5.92, 4.88, 5.51, and 5.59, respectively. When immune spleen cells were treated with anti-Thy 1.2 serum the PD50 value was increased from 4.73 to 6.09, i.e., protection was reduced greater than or equal to 95%. Similar treatment of immune thymic cells reduced protection below measureable values. Anti-B cell sera (anti-IgM and anti-Ly 4.2) did not reduce the protective effect of immune spleen or marrow cells. These results indicate that a major protective cell population in each of these compartments was theta-positive. Experiments were carried out to characterize the cortisone (CS) and x-ray sensitivity of immune spleen, thymic, and marrow cells .When donor mice were treated with 12.5 mg of cortisone acetate/day for 2 days before lymphoid cells were harvested, the orotective effects of immune spleen cells, but not immune thymic or marrow cells, was reduced. When immune spleen cells were x-irrated in vitro, their protective effect was reduced by 350 R and abolished by 1000 R. When mice received whole boyd x-irradiation 24 hr before immune spleen cells were transferred their protective effect was reduced by 1000 R but only slightly lowered by 350 R. The possible significance of the multicompartmental nature of immunity to leukemia was discussed.     

Antibody formation in mouse bone marrow. VIII. Dependence on potentially circulating memory cells: a study with parabiotic mice.

Mouse bone marrow barely contains antibody-producing plaque-forming cells (PFC) during the primary response to sheep red blood cells (SRBC). However, during the secondary response, the number of IgM, IgG, and IgA PFC in the bone marrow can rise to a level which surpasses the number of PFC in all the other lymphoid organs together. In the present paper we investigated whether the capacity of immune mice to react upon a booster injection of SRBC with a bone marrow PFC response can be transferred from immune to nonimmune mice. Therefore, mice primed with SRBC 6 months previously and nonprimed syngeneic mice were joined for parabiosis and were separated from each other at various intervals after joining. These separated mice were subsequently immunized with SRBC. It was found that, after 3 weeks of parabiosis, the nonprimed members reacted upon an injection of SRBC with a bone marrow IgM, IgG, and IgA PFC response as high as did the previously primed members. Furthermore it could be demonstrated by means of cell transfer experiments that, after a period of parabiosis of 3 weeks, the bone marrow and spleen of the normal mice contained about as many memory cells as the bone marrow and spleen of the immune mice. These results suggest that antibody formation in mouse bone marrow is dependent on a population of potentially circulating memory cells.     

Hybrid resistance to parental mast cell precursors in the skin of (WB X C57BL/6)F1-W/Wv mice

When bone marrow cells of (WB X C57BL/6)F1-+/+ (WBB6F1-+/+) and WB-+/+ (WB) mice were directly injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, mast cell clusters appeared at the injection sites. However, the number of WB bone marrow cells necessary for appearance of mast cell clusters was significantly larger than when bone marrow cells of WBB6F1-+/+ mice were used. When WB bone marrow cells were mixed either with WB thymus cells or with silica particles, the proportion of injection sites at which mast cell clusters appeared increased to the level that was observed after the injection of the same number of WBB6F1-+/+ bone marrow cells. When suckling WBB6F1-W/Wv mice of less than or equal to 18 days of age were used as recipients, bone marrow cells of WBB6F1-+/+ and WB mice produced mast cell clusters with a comparable efficiency. Both syngeneic thymus cells and silica particles are known to abrogate the hybrid resistance that is observed in the spleen against parental hematopoietic stem cells. The hybrid resistance in the spleen is not detectable in suckling mice, either. Thus, the poor growth of mast cell precursors in the skin and the poor growth of hematopoietic stem cells in the spleen seem to be regulated by the same mechanism.