Stromal cells in long-term cultures of liver, spleen, and bone marrow at different developmental ages have different capacities to maintain GM-CFC proliferation.

Measurements were made of the granulocyte-macrophage colony-forming cell (GM-CFC) yield in long-term cultures established from different combinations of stroma and hemopoietic recharge inocula derived from hemopoietic organs at different stages of their embryological development. Results indicated differences in the supporting capacity of the stroma, related to the hemopoietic activity of the organ of origin. Stroma derived from hemopoietically active organs (adult bone marrow, neonatal spleen, fetal liver) supported the proliferation of GM-CFC to a larger extent than stroma derived from organs with a low hemopoietic activity (neonatal bone marrow liver at 2 days; spleen at 3 weeks). Regardless of the origin of the hemopoietic cells, stroma from adult bone marrow displayed the highest ability to support GM-CFC proliferation. The capacity of GM-CFC from hemopoietic recharge cell populations to proliferate on stroma was not related to the hemopoietic activity of their organ of origin. Regardless of their organ of origin the GM-CFC present in each of the different hemopoietic recharge populations were able to proliferate provided that they were seeded on an appropriate stroma. These experiments showed that stromal cells cultured from hemopoietic organs at different developmental ages determine the hemopoietic activity of long-term cultures as measured via GM-CFC recovery.

Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.

Toxicity of 241Am in male C57BL mice: relative risk versus 226Ra.

A life-span study on male C57BL mice after injection of various doses of 241Am was conducted. The effects on life span were evaluated and the incidence of tumors was determined by procedures that take competing risks into account. Bone tumors were induced in the mice by injections of 22 and 58 Bq 241Am per g. The mice died early from nonneoplastic diseases at the higher dose levels (190, 373, and 1197 Bq 241Am/g). Additionally, spontaneously occurring tumors such as liver carcinomas, lymphosarcomas, and lymphoreticulosarcomas occurred at an enhanced rate with increasing dose level. The data for survival time after 241Am injection and death with bone tumor were compared to data collected previously for 226Ra-injected mice of the same C57BL strain. This enabled direct comparison in the same strain of the effects of the bone-surface seeker 241Am to the effects of the bone-volume seeker 226Ra. The proportional hazards model was applied and the rate of death with bone tumor was 12.9 +/- 5.2 times higher after 241Am injection than after 226Ra injection if the regression covariate was the average dose to the skeleton. The relative risk was 3.5 +/- 1.7 if regressed on the injected radioactivity. The mortality rate after 241Am injection was 20.4 +/- 3.6 times higher than after 226Ra injection if regressed on average dose to the skeleton.

Proliferation activity of stromal stem cells (CFU-f) from hemopoietic organs of pre- and postnatal mice.

Stromal stem cells (CFU-f assay) from hemopoietic organs of fetuses, in contrast to adult animals, exhibit a high proliferation activity. This implies that these CFU-f are radiosensitive and potential target cells after radioactive contamination of fetuses. Furthermore, the percentage of CFU-f in DNA synthesis is correlated with the hemopoietic activity in liver, spleen, and bone marrow. As hemopoiesis starts, high numbers of CFU-f are in S phase. In fetal liver, spleen, and bone marrow, values of 70, 43, and 58%, respectively, are reached. As hemopoietic activity decreases in liver and stabilizes in spleen and bone marrow, mitotic activity of these stromal stem cells becomes undetectable.

Protracted treatment of C57B1 mice with Zn-DTPA after 241Am injection reduces the long-term radiation effects.

Repeated intraperitoneal injections of ZnNa3-diethylenetriaminepenta-acetate (ZnDTPA), once a week, during 8 successive weeks, and starting 4 days after injection of 58 and 373 kBq 241Am/kg to C57B1 mice, were an effective protection against long-term radiation damage. At both dose levels of 241Am, Zn-DTPA reduced the 241Am concentration in bones by between 33% and 45%, and in the liver by 97%. Mean survival with 241Am was shortened in Zn-DTPA-treated mice, by 17% at the lower dose level and by 70% at the higher dose level. After treatment with DTPA at the lower 241Am level, survival became equal to that of control mice without 241Am, while at the higher level life span was still shortened. After the lower 241Am dose the incidence of bone tumours, liver carcinomas and the total number of all malignant tumours were significantly reduced by chelation therapy. The decrease in bone tumour incidence was proportional to the decrease in 241Am concentration and reduction in cumulative radiation dose in bone after chelation therapy. The incidence of liver carcinomas was reduced to that in non-241Am-injected mice and the reduction was thus proportional to the 97% reduction in 241Am concentration of the liver at the end of chelation therapy. After the higher 241Am dose no tumours showed up in sham-treated mice, probably due to the overkill effect on the cells at risk. In the corresponding Zn-DTPA-treated mice, bone tumours and a few other malignant tumours were observed.

Distribution of 241Am in offspring from BALB/c mice injected with 241Am at 14 days of gestation: relation to calcium and iron metabolism and comparison with distribution of 241Am after injection of adults.

Pregnant mice were given intravenous injections of 241Am citrate at 14 days of gestation. The fetal skeleton had a higher or similar uptake of 241Am per gram of fresh tissue than the liver. In comparison, the liver in adults concentrated 5 to 20 times more 241Am per gram of fresh tissue than the bones. Measurement of changes in calcium and iron content and concentration with time, showed that in the developing mice intensive calcification of bones determined the uptake of 241Am. The 241Am uptake was related to the calcium concentration of the fetal bones, which was greater at 14 days of gestation in the anterior bones, the mandibles and calvaria, than in the ribs and femurs. Transfer of 241Am to pups via milk resulted in further accumulation of 241Am in the skeleton and liver. The incorporation in the skeleton persisted after weaning and contributed to the lifetime body burden. The 241Am concentration decreased rapidly with time after injection in relation to the growth of the organs. Radiation dose rates and cumulative radiation doses were calculated for liver and bones of contaminated offspring.

Relative effectiveness of 241Am vs 226Ra approached by haemopoietic stem cell studies in various bone marrow sites of contaminated mice.

Haemopoietic stem cell assays allow investigation of local radiation damage at any time after contamination. These techniques were used to determine the relative effectiveness of 241Am and 226Ra in inducing damage in various cortical and trabecular bone marrow sites. Male Balb/c mice were injected with either 230 or 660 kBq 226RaCl2/kg body weight or with one of three doses of monomeric 241Am citrate at 138, 552 or 768 kBq 241Am/kg body weight. At 7 time intervals after injection (from 4 hr to 100 days) the colony-forming capacity of pluripotent stem cells (= CFU-s) and macrophage-granulocyte committed stem cells (CFU-c) was assayed in sternal marrow, marrow of lumbar vertebrae, of the trabecular distal end of the femur and of the femoral shaft (in which were distinguished axial marrow and marrow situated peripherally near the cortical bone). 226Ra retention and 241Am retention were measured in these bone sites. Skeletal doses and doses to the bone marrow sites were calculated. On the base of injected activity, 241Am was more effective than 226Ra in reducing the number of CFU-s and CFU-c but the effectiveness varied in the various bone marrow sites. CFU-s and CFU-c response is considered in relation to the retained radioactivity in the surrounding bone matrix and in relation to the mean marrow dose.

226Ra induced bone-cancers: the effects of a delayed Na-alginate treatment.

At the present time no unequivocal evidence exists which shows that a reduction in the body-burden of a radionuclide by decorporative treatment results in a proportional decrease in the risk of long-term radiation effects. We have investigated the effectiveness of the daily administration of Na-alginate via the diet in removing 226Ra from the skeleton and in reducing the number of late effects such as osteosarcomas. The animals used were male C57Bl mice which had been injected with one of three different amounts of 226Ra (4.4, 10.7 or 24.8 kBq) four days prior to the onset of the decorporative treatment. The results showed that although this treatment was able to produce a substantial reduction in the 226Ra content of the mice it did not reduce the incidence of osteosarcoma. These results question the effectiveness of decorporation procedures initiated at longer times after contamination.

Stromal stem cells (CFU-f) in yolk sac, liver, spleen and bone marrow of pre- and postnatal mice.

Our results demonstrate that in yolk sac, liver, spleen and femoral bone marrow of mice at ages ranging between 11 d of gestation and adult life, important changes in the stromal stem cell population (CFU-f assay) occur which are correlated with haemopoiesis. In the liver, spleen and bone marrow, high numbers of CFU-f precede high haemopoietic stem cell values. As haemopoiesis starts in the spleen, CFU-f numbers in fetal liver are low. Similarly, CFU-f numbers decrease in the spleen as bone marrow haemopoiesis starts. This suggests the existence of a migration stream of stromal stem cells. In spleen and bone marrow, CFU-f numbers decrease towards adult life as these organs maintain a stable haemopoietic activity.

Sparing of bone marrow stem cells by long-term administration of Na-alginate to 226Ra contaminated mice.

226Ra toxicity studies form the experimental basis for the estimation of radiation risk from internal emitters in man. We investigated whether treatment with Na-alginate is able to protect haemopoietic bone marrow cells against alpha-irradiation from 226Ra contamination. Doses from 4 to 14 micronCi/kg were injected intraperitoneally in mice 12 days before the start of the treatment. Damage to marrow stem cells was assessed by the exogene clonal spleen technique. Collection of marrow cells by two methods was compared. In the lower dose groups no influence on stem cell survival is noticed. but from 9.0 micronCi/kg a decrease in the number of surviving stem cells is observable in non treated animals. while in animals treated with Na-alginate fewer stem cells are damaged. These preliminary data agree with the hypothesis that Na-alginate stimulates removal of 226Ra mainly from the endosteal bone surfaces, reducing the local 226 Ra dose which accounts for damage to marrow stem cells within the range of alpha-rays at the endosteal surfaces.

Haemopoiesis in long-term cultures of liver, spleen and bone marrow of pre- and postnatal mice: CFU-GM production.

The CFU-GM yield in confluent long-term cultures (LTC) derived from liver, spleen and bone marrow cells at different gestational and postnatal ages has been studied after the stromal adherent layer reached confluency. The stromal cell compartment of fetal and neonatal haemopoietic organs is able to sustain haemopoiesis in vitro. Moreover, the granulocyte-macrophage stem cell (CFU-GM) yield of these LTC reflects the CFU-GM content of the haemopoietic organs from which the cultures are originated. LTC from the liver produce high numbers (between 100 and 150 CFU-GM per well) of CFU-GM if the cultures are derived from fetal livers between 13 d of gestation and birth. Cultures from spleens just before and after birth, give maximal CFU-GM numbers (between 50 and 100 CFU-GM per well). The CFU-GM yield in long-term bone marrow cultures increases 10 times from 17-day-old fetus towards adult life (between 700 and 1000 CFU-GM per well.