Radiosensitivity of cells-precursors of hemopoietic stroma (CFU-F) in the rat bone marrow under the effect of 60Co gamma irradiation in various conditions]

Conditions have been developed for cloning cells-precursors of rat bone marrow haemopoietic stroma, that form in culture dense and sparse fibroblast colonies (CFU-F) at a plating efficiency of 10(-4). Radiosensitivity of rat bone marrow CFU-F, with 60Co-gamma-irradiation in vitro, is characterized by the values of Do and n of 1.87 Gy and 1.4 respectively for all clones; 0.65 Gy and 6.7 for dense clones, and 4.27 Gy and 1.0 for sparse clones. This confirms the observed heterogeneity of CFU-F population consisting of highly radiosensitive and radioresistant subpopulations. The parameters of rat bone marrow CFU-F are nearly the same with irradiation both in vivo and in vitro; with in situ irradiation, the oxygen effect comes into play in a radiosensitive subpopulation of CFU-F; the OER values are 1.6, 2.6 and 0.9 for all, dense and sparse clones respectively.     

Radiosensitivity of clonogenic granulocytic macrophage cell precursors in the bone marrow and spleen of tumor-bearing mice

Clonogenic granulocytic macrophagal cells-precursors (CFU-DC) of bone marrow and spleen of intact C57Bl/6 mice and those inoculated subcutaneously with LLC tumor cells do not substantially differ in their radiosensitivity; the concentration of CFU-DC in the spleen markedly varies as tumor grows. The values of Do and extrapolation number n for CFU-DC of the bone marrow are 0.9-1.4 and 1.5-3.0 Gy, and of the spleen, 0.8-1.6 and 1.0-2.6 Gy, respectively.     

Effect of the U-2 fraction of splenic extract from Horsfield's terrapin on the hematopoiesis of mammals

Intraperitoneal administration of a spleen extract from Testudo horsfieldi and its U-2 fraction increases the number of endogenous splenic haemopoietic colonies. The U-2 fraction administered to irradiated (4 Gy) mice increases the number of bone marrow CFUs. Bone marrow cells of exposed (4 Gy) mice preincubated in vitro with the U-2 fraction also increase the number of exogenous colonies in the recipient's spleen.     

Radiosensitivity of pluripotent stem cells detectable by cloning in the spleen of irradiated mice

It was shown that the dose--effect curves describing the radiosensitivity of CFUc of the bone marrow irradiated in vitro (0.04-3.7 Gy) and treated with normal rabbit serum (NRS) and anti-mouse-brain serum (AMBS) has two differently sloping portions indicating that two CFUc populations differing in radiosensitivity are present in the bone marrow. D0 was 0.93 Gy after irradiation with doses of 0.04-0.75 Gy and treatment with NRS, and 0.33 Gy after incubation of the bone marrow with AMBS. The addition of thymus cells "straightened" the dose--effect curve for the bone marrow treated with AMBS: in this case D0 was 1.81 Gy exceeding considerably the values of D0 for intact bone marrow. The CFUc population is suggested to be heterogeneous in radiosensitivity.     

CFU-S and haematopoietic microenvironment in mice after fractionated irradiation. A study on spleen colonies.

The paper is aimed at evaluating the quantity and quality of the haematopoietic stem cells, CFU-S, in the bone marrow and the functional effectiveness of the haematopoietic microenvironment of the spleen in two time intervals after repeated exposure of mice to doses of 0.5 Gy gamma-rays once a week (total doses of 12 and 24 Gy). After irradiation, bone marrow was cross-transplanted between fractionatedly irradiated and control mice. The parameter evaluated were numbers of spleen colonies classified into size categories. The data obtained provide evidence for a significant damage to the CFU-S, concerning both their number and proliferation ability, after both total doses used. The functional effectiveness of the haematopoietic microenvironment of the spleen was impaired only in bone marrow recipients receiving a transplant after having been exposed to a total dose of 24 Gy; this dose combined with subsequent pre-transplantation irradiation resulted in a marked suppression of cell production within the spleen colonies formed from a normal bone marrow on the spleens of fractionatedly irradiated mice.     

Local regulation of haemopoietic stem cell proliferation in mice following irradiation

Changes in the kinetic state of pluripotent haemopoietic spleen colony forming cells (CFU-S) and of the CFU-S proliferation stimulator have been studied following whole-body X-irradiation. Rapid recruitment of CFU-S into cell cycle by 30 min after irradiation was observed following low doses (0.5 Gy) but a delay of 6 h occurred after higher doses (1.5 and 4.5 Gy). These changes in proliferative state correlated with the presence of the CFU-S proliferation stimulator. CFU-S irradiated in vitro in bone marrow plugs were also recruited into cycle illustrating directly the local nature of the feedback mechanism. CFU-S removed from 1.5 Gy irradiated recipients at a time when they were not in cycle were not responsive to the CFU-S proliferation stimulator. The CFU-S proliferation stimulator was produced by Ia positive cells in the irradiated bone marrow. The regulation changes occurring shortly after irradiation cannot simply be controlled by the size of the CFU-S compartment.     

Survival of erythroid burst-forming units and erythroid colony-forming units in canine bone marrow cells exposed in vitro to 1 MeV neutron radiation or X rays

Conditioned media (CM) from allogeneic stimulated cultures of light density cells (less than 1.08 g/cm3) from the peripheral blood of normal dogs were used to stimulate the growth of erythroid burst-forming units (BFU-E) in bone marrow from normal dogs. Maximum numbers of BFU-E were obtained when 5% (vol/vol) 3 X CM and 2 U/ml erythropoietin were added to plasma clot cultures of bone marrow cells. In addition, the radiation sensitivity (D0 value) was determined for CFU-E and for BFU-E in bone marrow cells exposed in vitro to 1 MeV fission neutron radiation or 250 kVp X rays. BFU-E were more sensitive than CFU-E to the lethal effects of both types of radiation. For bone marrow cells exposed to 1 MeV neutron radiation, the D0 for CFU-E was 0.27 +/- 0.01 Gy, and the D0 for BFU-E was 0.16 +/- 0.03 Gy. D0 values for CFU-E and BFU-E were, respectively, 0.61 +/- 0.05 Gy and 0.26 +/- 0.09 Gy for cells exposed to X rays. The neutron RBE values for the culture conditions described were 2.3 +/- 0.01 for CFU-E and 1.6 +/- 0.40 for BFU-E.     

Haemopoiesis in the beagle foetus after in utero irradiation

On day 33 of gestation, foetal beagles were irradiated in utero (0.9 Gy of 60Co gamma-irradiation, 0.4 Gy/min). Foetal haematocytopoiesis was studied during the third trimester of gestation (days 42-55). Peripheral blood nucleated cell counts were 33 per cent lower than normal on day 44 and continued to be lower until day 49, when values became higher than normal. Splenic cellularities of irradiated pups on day 44 were more than 3 times those of the nonirradiated, but thereafter they were similar to normal. Differences in haemopoietic progenitor cell activity between irradiated and normal foetuses were observed. In comparison with the other foetal tissues, the foetal liver appeared to experience greater radiation injury. For example, on day 44, the irradiated liver BFU-E, CFU-E, and GM-CFC per 10(5) cells were almost fivefold lower than normal values. Spleens of irradiated foetal beagles contained a marked increase in all haemopoietic progenitor cells (BFU-E, CFU-E, and GM-CFC) and recognizable proliferative granulocytic cells and nucleated erythroid cells. The haemopoietic activity of the irradiated bone marrow during days 42-44 was similar to that of the irradiated spleen, and compensated for the damaged liver. However, unlike the irradiated spleen, the irradiated bone marrow had decreased BFU-E activity compared with the values for the nonirradiated bone marrow during days 48-55. Until day 50, the irradiated marrow contained fewer recognizable proliferative granulocytic cells but more nucleated erythroid cells.     

Interleukin-2 stimulates the colony-forming activity of in vitro irradiated bone marrow

The data are presented on the stimulatory effect of interleukin-2 on the formation of spleen exocolonies from bone marrow irradiated in vitro with doses of 0.5 to 2.5 Gy. It is suggested that the effect observed is associated with the increased proliferation of CFUs survived after irradiation.     

Local regulation of haemopoietic stem cell proliferation in mice following irradiation

Abstract Changes in the kinetic state of pluripotent haemopoietic spleen colony forming cells (CFU-S) and of the CFU-S proliferation stimulator have been studied following whole-body X-irradiation. Rapid recruitment of CFU-S into cell cycle by 30 min after irradiation was observed following low doses (0.5 Gy) but a delay of 6 h occurred after higher doses (1.5 and 4.5 Gy). These changes in proliferative state correlated with the presence of the CFU-S proliferation stimulator. CFU-S irradiated in vitro in bone marrow plugs were also recruited into cycle illustrating directly the local nature of the feedback mechanism. CFU-S removed from 1.5 Gy irradiated recipients at a time when they were not in cycle were not responsive to the CFU-S proliferation stimulator. The CFU-S proliferation stimulator was produced by Ia positive cells in the irradiated bone marrow. The regulation changes occurring shortly after irradiation cannot simply be controlled by the size of the CFU-S compartment.     

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.     

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.     

Recovery of hematopoiesis in mice following a continuous irradiation with a dose rate of 0.957 Gy/d and a total accumulated dose of 19.14 Gy. I. Bone marrow, spleen and thymus gland

This paper deals with the evaluation of histological changes in the bone marrow, spleen and thymus of mice after continuous irradiation with a dose rate of 0.957 Gy/day and a total accumulated dose of 19.14 Gy. Erythropoiesis in the spleen could be recovered quickly, significantly exceeding the spleen erythropoiesis of the controls on the seventh post-irradiation day. Myelopoiesis in the bone marrow could be recovered until the 21st day and erythropoiesis until the 28th day after the end of irradiation. Lymphopoiesis in the thymus could be recovered on the 28th day approximately and in the spleen roughly on the 60th day after the end of irradiation.     

Effect of pre-exposure to beta rays of tritium on some biochemical parameters measured in organs of rats subsequently irradiated with fast neutrons

The experiment examined biological responses produced by combined sequential exposure to low-level tritium contamination, followed by challenging irradiation with fast neutrons. Modifications of endogenous antioxidant potential of different organs in rats were discussed in relation to tissue radiosensitivity. Rats pre-contaminated to 7 cGy and 35 cGy have been additionally irradiated to 1 Gy with fast neutrons. Lipid peroxide level was determined in liver, kidney, small intestine, spleen, bone marrow, and plasma. Reduced glutathione (GSH) level and glucose-6-phosphate dehydrogenase (G6PDH) activity were determined in erythrocytes. An in vitro thymidine uptake assay was performed in isolated bone marrow cells. The lipid peroxide level decreased significantly only in liver and kidney from rats pre-exposed to 35 cGy. For small intestine and spleen, tissues of comparatively higher radiosensitivity, no induced radioprotection was observed, as reflected in the homeostasis of the lipid peroxides. The same behavior was observed in bone marrow, the most radiosensitive tissue studied. However, the bone marrow thymidine-incorporation assay revealed a possible adaptive-type reaction in rats pre-exposed to 35 cGy. We conclude that for radiosensitive tissues pre-exposure to chronic low doses of low linear energy transfer (LET) irradiation has no protective effect on their antioxidant status, whereas a protective effect is observed in radioresistent tissues.     

The possible pathways of IL-2 stimulation of colony formation by bone marrow cells irradiated in vitro

The authors have made an attempt to find out the reasons of IL-2 stimulation of spleen colony growth by in vitro (1 Gy) irradiated bone marrow cells. It was shown that the effect of IL on haemopoiesis manifests itself with merely small radiation doses implying that the influence of the preparation makes the process of haemopoietic organ repopulation start at a higher level of cell survival, which is presumably related to a more active repair of radiation-induced CFUs damages: this leads, with other things being equal (e.g. proliferation rate and f factor), to a higher yield of colonies than it is observed with the recipients protected with the exposed bone marrow only.     

Protective effect of sesamol against 60Co γ-ray-induced hematopoietic and gastrointestinal injury in C57BL/6 male mice

Abstract

Protection of γ-ray-induced injury in hematopoietic and gastrointestinal (GI) systems is the rationale behind developing radioprotectors. The objective of this study, therefore, was to investigate the radioprotective efficacy and mechanisms underlying sesamol in amelioration of γ-ray-induced hematopoietic and GI injury in mice. C57BL/6 male mice were pre-treated with a single dose (100 or 50 mg/kg, 30 min prior) of sesamol through the intraperitoneal route and exposed to LD_50/30 (7.5 Gy) and sublethal (5 Gy) dose of γ-radiation. Thirty-day survival against 7.5 Gy was monitored. Sesamol (100 mg/kg) pre-treatment reduced radiation-induced mortality and resulted survival of about 100% against 7.5 Gy of γ-irradiation. Whole-body irradiation drastically depleted hematopoietic progenitor stem cells in bone marrow, B cells, T cell subpopulations, and splenocyte proliferation in the spleen on day 4, which were significantly protected in sesamol pre-treated mice. This was associated with a decrease of radiation-induced micronuclei (MN) and apoptosis in bone marrow and spleen, respectively. Sesamol pre-treatment inhibited lipid peroxidation, translocation of gut bacteria to spleen, liver, and kidney, and enhanced regeneration of crypt cells in the GI system. In addition, sesamol pre-treatment reduced the radiation-induced pattern of expression of p53 and Bax apoptotic proteins in the bone marrow, spleen, and GI. This reduction in apoptotic proteins was associated with the increased anti-apoptotic-Bcl-x and PCNA proteins. Further, assessment of antioxidant capacity using ABTS and DPPH assays revealed that sesamol treatment alleviated total antioxidant capacity in spleen and GI tissue. In conclusion, the results of the present study suggested that sesamol as a single prophylactic dose protects hematopoietic and GI systems against γ-radiation-induced injury in mice.     

Determination of the radiosensitivity of interphase-dying cells in the thymus, spleen and bone marrow of rats by flow cytometry

The flow cytofluorometry of cells stained with a DNA-specific probe was used to determine the share of dying cells (containing less than 2C DNA) in thymus, spleen and bone marrow cells of irradiated rats. The cell death curves for spleen and bone marrow had a plateau by the 6th h, and for thymus, by the 10th h following irradiation with different doses. On the basis of the dose-response relationship the share of cells dying in the interphase was determined in each organ under study, and dose-response curves shaped. All the curves had no shoulder. Do was 3.0, 3.0 and 3.7 Gy for thymus, spleen, and bone marrow cells, respectively.     

THE PROLIFERATIVE CAPACITY OF HAEMOPOIETIC COLONY FORMING UNITS IN THE RAT

After transplantation into rats lethally treated with cytotoxic chemicals both bone marrow and spleen CFU in the spleen and spleen derived CFU in the bone marrow expand with doubling times ( T _d) of approximately 18 hr. However, bone marrow derived CFU in the bone marrow have a T _d of 36 hr. Evidence obtained using tritiated thymidine in vitro and methotrexate in vivo show that the proliferation rate of bone marrow derived CFU is similar in both the bone marrow and spleen and calculations suggest that the different T _d between these two sites is due to the higher loss of CFU through differentiation in the bone marrow compared to the spleen. These findings further support the hypothesis of an environment in the spleen which favours CFU self-maintenance over differentiation with the opposite situation occurring in the bone marrow.     

小鼠低剂量辐射损伤模型的初步研究

目的:对小鼠低剂量辐射损伤模型进行初步研究并筛选敏感检测指标.方法:实验分7组,1组为正常组,其余6组用XHA600直线加速器射线照射,每组取一部分小鼠于照射后2、4、8、16h测定红细胞(RBC)、白细胞(WBC)、血小板(PLT)和血红蛋白(HGB)含量;测定小鼠肝脏和脾脏重量及其组织中的超氧化合物歧化酶(SOD)和丙二醛(MDA)含量;剩余小鼠于4周后观察骨髓切片中血细胞变化.结果:照射后4h测定血常规,发现累积辐射0.4Gy组小鼠RBC有降低趋势,WBC、HGB显著降低,PLT显著升高;肝脏、脾脏的湿质量显著降低;小鼠肝脏组织中MDA的含量显著升高、SOD的活力显著降低;4周后小鼠骨髓细胞的病理改变与单次照射剂量相关,单次较大剂量(如0.6Gy)照射对骨髓细胞影响较大,在4周观察期不能自身恢复,而多次累积照射对骨髓细胞病理改变较小.结论:小鼠低剂量辐射损伤模型的最佳造模剂量为累积照射0.4Gy即每次照射100mGy,间隔一天,连续照射4次.     

Sister-chromatid exchange studies on direct- and indirect-acting clastogens in mouse primary cell cultures

An in vitro sister-chromatid exchange (SCE) assay using mouse primary bone marrow and spleen cells was conducted with both direct- and indirect-acting genotoxic agents. 2,4,7-Trinitrofluorenone, a direct-acting genotoxic agent, induced a significant dose-related increase in SCEs. In both bone marrow and spleen cells, 2.0 micrograms/ml caused an approx. 3-fold increase in SCE level over control values. Cyclophosphamide, an indirect-acting genotoxicant which requires metabolic activation for its clastogenicity, induced a significant increase in SCEs in the presence of S9 from liver of rats pretreated with Aroclor-1254. A dose of 2 micrograms/ml resulted in a 2-fold increase in bone marrow and a greater than 5-fold increase in spleen cells. Benzo[a]pyrene, another indirect-acting genotoxicant, also induced significant dose-related SCE responses in both cell types. It seems that primary bone marrow and spleen cell culture systems can detect both direct- and indirect-acting genotoxicants and may be useful for routine and/or comparative cytogenetic studies.