Lifetime treatment of mice with azidothymidine (AZT) produces myelodysplasia.

AZT has induced a macrocytic anemia in AIDS patients on long term AZT therapy. It is generally assumed that DNA elongation is stopped by the insertion of AZT into the chain in place of thymidine thus preventing the phosphate hydroxyl linkages and therefore suppresses hemopoietic progenitor cell proliferation in an early stage of differentiation. CBA/Ca male mice started on AZT 0.75 mg/ml H2O at 84 days of age and kept on it for 687 days when dosage reduced to 0.5 mg/ml H2O for a group, another group removed from AZT to see recovery, and third group remained on 0.75 mg. At 687 days mice that had been on 0.75 mg had average platelet counts of 2.5 x 10(6). Histological examination on 9 of 10 mice with such thrombocytopenia showed changes compatible with myelodysplastic syndrome (MDS). A variety of histological patterns was observed. There were two cases of hypocellular myelodysplasia, two cases of hypersegmented myelodysplastic granulocytosis, two cases of hypercellular marrow with abnormal megakaryocytes with bizarre nuclei, one case of megakaryocytic myelosis associated with a hyperplastic marrow, dysmyelopoiesis and a hypocellular marrow and two cases of myelodysplasia with dyserythropoiesis, hemosiderosis and a hypocellular marrow. Above mentioned AZT incorporation may have induced an ineffective hemopoiesis in the primitive hemopoietic progenitor cells, which is known to be seen commonly in the myelodysplastic syndrome.

Survival of spleen colony-forming units (CFU-S) of irradiated bone marrow cells in mice: evidence for the existence of a radioresistant subfraction.

Because of increasing evidence of heterogeneity in the hematopoietic stem cell compartments, the radiosensitivity of spleen colony-forming units (CFU-S) was reevaluated to ascertain whether the classical single exponential curve for a graded dose of radiation is applicable at higher doses of radiation, 400-600 cGy. Bone marrow cells (BMC) removed from mice immediately after death under anesthesia were irradiated in vitro. Great care was taken to exclude anoxic effects during irradiation and to avoid any possible effects in the recipient mice from injection of excessive numbers of BMC. By estimating the number of cells to be injected to produce numbers of colonies within the evaluation range of the assay, we obtained a radiation survival curve that appeared to have a multiphasic concave shape; the D0 value for the 400-600 cGy range was estimated to be about 275 cGy, whereas the D0 for the lower doses was 95 cGy, the same value as previously reported. The reason a single exponential survival curve was previously obtained after graded doses of radiation is discussed, and a comparison of those results with the present data from in vitro radiation is made. Lacking experimental evidence, we speculate that the major factor that determines the slope of the survival curve is the degree to which the stem cells are in their normal hematopoietic environment during the irradiation. The probable existence of a fraction surviving after an exposure to 600 cGy, estimated by the limiting dilution assay, was about 1 per 2 x 10(6) BMC. Such radio-insensitive CFU-S appear to be primitive CFU-S, which can contribute materially to the long-term survival of lethally irradiated bone marrow recipients.

Hypermutability of mouse chromosome 2 during the development of x-ray-induced murine myeloid leukemia.

In an effort to identify the precise role of a deletion at regions D-E of mouse chromosome 2 [del2(D-E)] during the development of radiation-induced myeloid leukemia, we conducted a serial sacrifice study in which metaphase chromosomes were examined by the G-banding technique. Such metaphase cells were collected from x-irradiated mice during the period of transformation of some of the normal hematopoietic cells to the fully developed leukemic phenotype. A group of 250 CBA/Ca male mice (10-12 weeks old) were exposed to a single dose of 2 Gy of 250-kilovolt-peak x-rays; 42 age-matched male mice served as controls. Groups of randomly selected mice were sacrificed at 20 hr, 1 week, and then at intervals of 3 months up to 24 months after x-irradiation. Slides for cytogenetic, hematological, and histological examination were prepared for each animal at each sacrifice time. An expansion of cells with lesions on one copy of chromosome 2 was evident in 20-25% of treated mice at each sacrifice time. The majority of such lesions were translocations at 2F or 2H, strongly suggesting hypermutability of these sites on mouse chromosome 2. No lesions were found in control mice. The finding leads to the possibility that genomic lesions close to 2D and 2E are aberrants associated with radiation leukemogenesis, whereas a single clone of cells with a del2(D-E) may lead directly to overt leukemia. The data also indicate that leukemic transformation arises from the cumulative effects of multiple genetic events on chromosome 2, reinforcing the thesis that multiple steps of mutation occur in the pathogenesis of cancer.

Influence of radiation fractionation on survival of mice and spleen colony-forming units.

C57Bl/6 mice were given 10 Gy X rays fractionated in several ways. There was a cyclical pattern of animal survival which was correlated to the fractionation interval and which indicated a periodicity of 6 h. Ten grays given in a single dose is fatal to 100% of the mice and depresses the CFU-S to about one per leg with no evidence of proliferation during the remaining life. Ten grays given in 2.5-Gy increments at 24-h intervals causes no fatalities and results in a similar CFU-S depression but is followed by an exponential increase in CFU-S over the ensuing 12 days. Although bone marrow from survivors of such treatment was comparable to control marrow in its capacity for short-term rescue, it was clearly inferior in its capacity for long-term rescue. The periodicity of 6 h suggests that the cells responsible for survival of the mice have been synchronized into more or less radiosensitive and radioresistant stages of the cell cycle as a result of the time between the 2.5-Gy increments. Implications for the CFU-S and long-term repopulating cells are discussed.

Anemia induced in splenectomized mice by administration of rhG-CSF.

Normal and splenectomized mice (SPLXM) were given rhG-CSF for 10 to 128 days and serial observations were made on blood counts for 128 days. After 10 days, mice were killed for histologic studies. All treatment schedules produced, in addition to elevated white blood counts, a macrocytic anemia which only partially responded to large doses of Epo. Stopping rhG-CSF treatment for 2 days resulted in the return of granulocytes, lymphocytes, monocytes, platelets and polychromatophilic erythrocytes to near normal levels, indicating a need for the continued presence of rhG-CSF to maintain peripheral blood increases. Treatment of normal and SPLXM with rhG-CSF induced marked granulocytic hyperplasia of the bone marrow with expansion of the granulocytic marrow into the adjoining muscle as in acute myelocytic leukemia. The hyperplasia is greater in the SPLXM than in the normal mouse where splenic hyperplasia occurs in all cell lines. The rhG-CSF also results in expansion of granulopoiesis into the normally fatty tail bone marrow in SPLXM. The rhG-CSF treatment produced marked increases in the assayable numbers of GM-CFU, G-CFU and M-CFU. The significance and mechanisms of induction of these changes are not clear. It is speculated that treatment with rhG-CSF has multicellular effects, suggesting that it initiates a cascade of molecular reactions that cause the effects observed.

Hematotoxicity and carcinogenicity of inhaled benzene.

CBA/Ca male mice have been exposed to benzene in air at 10, 25, 100, 300, 400, and 3000 ppm for variable intervals 6 hr/day, 5 days/week for up to 16 weeks. Two weeks of inhaling 10 ppm produced no hematologic effects; 25 ppm induced a significant lymphopenia. Inhalation of 100, 300, and 400 ppm produced dose-dependent decreases in blood lymphocytes, bone marrow cellularity, marrow content of spleen colony-forming units (CFU-S) and an increased fraction of CFU-S in DNA synthesis. Exposure of mice to 300 ppm for 2, 4, 8, and 16 weeks produced severe lymphopenia and decrease in marrow CFU-S. Recovery was rapid and complete after 2 and 4 weeks of exposure. After 8 and 16 weeks of exposure, recovery of lymphocytes was complete within 8 weeks. It took 16 weeks for the CFU-S to recover to that of the age-matched controls after 8 weeks of exposure and 25 weeks to recover to age-matched after 16 weeks of exposure. Inhalation of 3000 ppm for 8 days was less damaging than inhalation of 300 ppm for 80 days (same integral amount of benzene inhaled). The inhalation of 3000 ppm has not increased the incidence of leukemia or shortened its latency for development. Inhalation of 300 ppm 6 hr/day for 16 weeks significantly increases the incidence of myelogenous neoplasms in male CBA/Ca mice. Inhalation of 100 ppm for same interval does not influence incidence of myelogenous neoplasms but does increase incidence of solid neoplasms particularly in female CBA/Ca mice. Benzene is a potent carcinogen in CBA/Ca mice.

Bone marrow cells other than stem cells seed the bone marrow after rescue transfusion of fatally irradiated mice.

In a previous publication, iodinated deoxyuridine (125IUdR) incorporation data were interpreted as indicating that spleen colony-forming units (CFU-S) in DNA synthesis preferentially seeded bone marrow. In the present studies, the CFU-S content of marrow from irradiated, bone-marrow transfused mice was directly determined. Pretreatment of the transfused cells with cytocidal tritiated thymidine resulted in an insignificant diminution in CFU-S content when compared with nontritiated thymidine pretreatment, implying that there is no preferential seeding. The 125IUdR incorporation data have been reinterpreted as being a result of the proliferation of other progenitor cells present that have seeded the bone marrow.

Effects of low level radiation upon the hematopoietic stem cell: implications for leukemogenesis.

These studies have addressed firstly the effect of single small doses of x-rays upon murine hematopoietic stem cells to obtain a better estimate of the Dq. It is small, of the order of 20 rad. Secondly, a dose fractionation schedule that does not kill or perturb the kinetics of hemopoietic cell proliferation was sought in order to investigate the leukemogenic potential of low level radiation upon an unperturbed hemopoietic system. Doses used by others in past radiation leukemogenesis studies clearly perturb hemopoiesis and kill a detectable fraction of stem cells. The studies reported herein show that 1.25 rad every day decrease the CFU-S content of bone marrow by the time 80 rads are accumulated. Higher daily doses as used in published studies on radiation leukemogenesis produce greater effects. Studies on the effect of 0.5, 1.0, 2.0, and 3.0 rad 3 times per week are under way. Two rad 3 times per week produced a modest decrease in CFU-S content of bone marrow after an accumulation of 68 rad. With 3.0 rad 3 times per week an accumulation of 102 rad produced a significant decrease in CFU-S content of bone marrow. Dose fractionation at 0.5 and 1.0 rad 3 times per week has not produced a CFU-S depression after accumulation of 17 and 34 rad. Radiation leukemogenesis studies published to date have utilized single doses and chronic exposure schedules that probably have significantly perturbed the kinetics of hematopoietic stem cells. Whether radiation will produce leukemia in animal models with dose schedules that do not perturb kinetics of hematopoietic stem cells remains to be seen.

Marrow transfusions increase pluripotential stem cells in normal hosts.

In earlier investigations of marrow transfusions into isogeneic, nonirradiated mice, the percentage of donor cells in the recipients' marrow and peripheral blood was found to vary between 16% and 40% following transfusion of 200 million marrow cells. The present experiments demonstrated that the hosts' pluripotential stem cells were elevated to an average of 132% of simultaneously assayed nontransfused controls. The elevation persisted for two months and then returned to normal. The similar magnitude of elevation of pluripotential stem cells and of the percentage of donor cells in the recipients indicates that the seeded stem cells did not replace the hosts' own, but were added to the existing complement of pluripotential cells. Implications for the regulation of stem cell numbers are discussed.

Benzene hematotoxicity and leukemogenesis.

Eight-to-twelve-week-old male and female C57B1/6 BNL mice were exposed to air or benzene vapor in air at a concentration of 10, 25, 100, 300, or 400 ppm. Benzene at concentrations of 100 ppm or higher for 10 exposures of 6 hours per day 5 days a week produced a reduction in bone marrow cellularity and the number of pluripotent stem cells in the bone marrow. The fraction of stem cells in DNA synthesis was also increased. Exposure to 300 ppm 6 hours a day 5 days a week for 2, 4, 8, and 16 weeks produced a diminution in the stem cell levels in bone marrow which returned to those of controls 2 weeks after benzene exposure for 2 and 4 weeks, 16 weeks after exposure for 8 weeks, and to 92% of controls 25 weeks after 16 weeks of exposure. There was a more rapid return of blood lymphocytes to the control level. Mice exposed to 300 ppm for 6 hours/day, 5 days per week for 16 weeks began dying at 330 days of age, whereas no deaths were observed in sham-exposed mice until 440 days of age. The benzene-exposed mice died in two waves: the first was from 330-390 days of age, with a second wave commencing at 570 days of age. The first wave of mortality was due primarily to thymic lymphomata. The second wave was due to a mixture of nonthymic lymphomata and solid tumors.

Relationship between number of spleen colonies and 125IdUrd incorporation into spleen and femur.

Graded numbers of bone marrow (BM) cells were injected into fatally irradiated mice. Eight days later the mice were given 3.0 microCi (1 Ci = 3.7 X 10(10) Bq) of 125IdUrd to label proliferating cells in the spleen and BM. On day 9 the mice were killed and the spleens and femurs were removed for splenic colony assay and measurement of radioactivity in the spleen and femurs. The number of splenic colonies shows a linear relationship with dose of marrow cells injected from 10(4) to 10(5) cells. The slope of the curve of spleen colonies versus number of cells injected is less than 1, implying that the fraction seeded in spleen decreases with number of cells injected. Above 10(5) and below 10(4) there is a striking departure from the simple linearity. Below 2 X 10(3) cells injected, the logarithm of the observed colony yield is linear with logarithm of the number of cells injected. Poisson calculation of the average number of pluripotent stem cells that should be present with numbers of marrow cells injected below 2 X 10(3) followed closely the actual observations. The data show that there is no detectible proliferation in the BM until the dose of marrow cells exceeds 3.5 X 10(4) cells. Induction of cells into cycle increases the seeding into the BM, and thymidine cytocide drastically reduces seeding in the BM, leading us to conclude that the BM is repopulated almost exclusively by stem cells in DNA synthesis.

Effects of benzene inhalation on murine pluripotent stem cells.

Effects of benzene inhalation on mouse pluripotent hematopoietic stem cells have been evaluated. Male mice 8--12 wk old were exposed to 400 ppm benzene for 6 h/d, 5 d/wk, for up to 9 1/2 wk. At various time intervals exposed and control animals were killed, and cardiac blood was evaluated for changes in white blood cell (WBC) and red blood cell (RBC) content. In addition, femora and tibiae were evaluated for total marrow cellularity, stem cell content (as measured by the spleen colony technique), and the percent of stem cells in DNA synthesis (as determined by the tritiated thymidine cytocide technique). Exogenous spleen colonies grown from marrow of exposed animals were counted, identified, and scored by histological type. Exposure to benzene caused significant depressions of RBCs and WBCs throughout the exposure period, which continued for at least 14 d after exposure. Bone marrow cellularity and stem cell content were also depressed in exposed animals throughout the study. Tritiated thymidine cytocide of spleen colony-forming cells was generally increased in exposed animals, perhaps indicating a compensatory response to the reduction of circulating cells. Spleen colonies of all types were depressed after exposure to benzene. The significance of the reduction in cellularity, stem cell content, and changes in morphology of spleen colonies is discussed in relation to cellular toxicity and residual injury.