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DYSFUNCTION AND NEOPLASIAS OF HAEMOPOIETIC AND OSTEOGENIC TISSUE FOLLOWING EXTERNAL IRRADIATION OR BONE-SEEKING RADIONUCLIDE CONTAMINATION IN UTERO OR DURING NEONATAL

Objective

The aims of the contract are to determine: .SP 0 the most radiosensitive period during prenatal and postnatal development for both the response of stromal and haemopoietic marrow cells and for the dysfunction or induction of neoplastic change in bone and bone marrow following contamination with alpha-particle emitting radionuclides or comparable doses of external low LET irradiation;
the functional quality of mature cells generated from haemopoietic tissue damaged by such radiations;
the identity and location of the sensitive cell populations;
the role of haemopoietic growth factors in the regulation and recovery of irradiation damaged tissue;
the features of the stromal populations identified by cellular and molecular techniques.

MRC Radiobiology Unit, Chilton.

The initial aims of the project are to observe the late effects of 32, 64 and 128 Bq/g 239Pu injected into pregnant CBA/H mice on days 4 and 13 of gestation. Only the male offspring will be kept and a total of 200 mice will be entered into each group. Subsequently, following investigations carried out on the diaplacental transfer and effects of 224Ra on haemopoiesis in CBA/H mice, a similar experiment is planned for the late effects of 224Ra also in CBA/H mice.

SCK/CEN, Mol.

Research tasks are the identification of the radiosensitive developmental period after 241Am contamination of Balb/c mice; and the investigation of the cellular and molecular origin of residual radiation damage in marrow after low doses with emphasis on stromal cells.

Paterson Institute, Manchester.

Investigations into:
the placental transfer of radionuclides and their distribution in foetal and neonatal tissues following acute or chronic administration at various phases of pregnancy and infancy (in close collaboration with MRC Radiobiology Unit, Chilton);
the long term damage to the haemopoietic progenitor cells in terms of total number, proliferative activity and self renewal capacity after radionuclide contamination or after homogeneous low LET irradiation given at different dose rates;
resulting long term disturbances in the spatial distributions of haemopoietic progenitor and stromal cells in the marrow spaces; .SP 0 the long-term damage to the haemopoietic microenvironment (using marrow culture techniques) and its capacity to generate a bone capsule supporting haemopoiesis under the renal capsule;
the manipulation of cell production using growth factors.

Radiobiological Institute, TNO, Rijswijk.

Stem cells, purified from mouse bone marrow (young and adult) and from mouse foetal liver will be cultured on stromal layers (mostly in collaboration with SCK/CEN, Mol) where the interaction between the purified stem cells and the stromal cells will be analyzed by the techniques of molecular biology.

Using similar methods to those used to purify stem cells, attempts will be made to identify and isolate the precursors of the cultured stromal cell layer. In collaboration with SCK/CEN, Mol, the growth kinetics and clonogenicity of these precursors will be characterized in order to evaluate the replacement of stromal cells after radiation damage.

Fluorescent in situ hybridization methods will be developed to detect which cells in the stromal layers are producing haemopoietic growth factors and to what extent that production can directly or indirectly be damaged by radiation.

Universidad Complutense de Madrid, Madrid.

Our contribution in this project will be focused on the biochemical characterization of functional deficiencies induced by radiation. Residual damage will be assessed by:
functional damage of mature granulocytes (phagocytosis dysfunction);
metabolic and energetic status of the stromal cells and their ability to synthesize matrix molecules.

The studies will be carried out in mice of different ages, including newborn, young and adults irradiated with single and repeated doses of external low LET irradiation or contaminated with alpha-particle emitting radionuclides in collaboration with Paterson Institute, Manchester, MRC Radiobiology Unit, Chilton and CIEMAT, Madrid. We will establish LTBMC's at different postirradiation times.

CIEMAT, Madrid.

Residual haemopoietic damage induced by irradiation of newborn and adult mice will be analyzed in terms of stem cell proliferation and differentiation. Total number, proliferative activity and self renewal capacity of the haemopoietic stem and progenitor cells will be evaluated.

Those irradiation protocols (sublethal low LET external irradiation or contamination with radionuclides) that induce residual damage in the progenitor populations will be used to determine also the effects on stem cell differentiation.
A long term experiment on CBA/H mice demonstrating the late effects of plutonium-239 administered in utero has begun by introducing into the experiment approximately 1000 male offspring from CBA/H females given 16 Bq g{-1}, 32 Bq g{-1} or 64 Bq g{-1} plutonium-239 on either day 4 or day 13 of gestation. To date only 5 animals are dead out of 953 introduced; no significant pathology has been seen.

In order to identify a radiosensitive stage in development, Balb/c mice have been radiocontaminated with americium-241 at different developmental ages and in different ways. Long term cultures (LTC) allow the in vitro study of radiation induced defects to haemopoietic precursor cells and to their microenvironment. Cellular and extracellular matrix components in these cultures have been analysed to find changes related to persistent haemopoietic dysfunction after in utero contamination with americium-241. The haemopoietic capacity of the stromal layer was evaluated by reseeding the stroma with haemopoietic pluripotent stem cells.

With respect to the cellularity and number of haemopoietic and stromal stem cells, neither the bone marrow cellularity nor the number of pluripotent haemopoietic stem cells were changed in the offspring after the different ways of in utero contamination.
The amount of progenitor cells for granulocytes and macrophages tended to decrease, while in contrast the number of stromal stem cells had the tendency to increase.
In the long term bone marrow cultures, at 13 weeks postpartum no differences were seen in the haemopoietic activity of LTC, which reflect the functional capacity of stromal cells to maintain haemopoiesis in vitro from either control mice or contaminated mice. At 24 weeks postpartum the haemopoietic activity of LTC was significantly diminished. At 32 weeks postnatally, the colony forming unit in granulocytes and macrophages (CFU-GM) yield in LTC derived from offspring contaminated in utero using osmotic pumps either between the 7th and 14th day of gestation or between the 14th and 19th day of gestation was significantly lower than that in control mice.
The cumulative dose to the femur in offspring reared by their own contaminated mother reached 1.7 cGy at 13 weeks postcontamination, 2.6 cGy 24 weeks after contamination and 3.3 cGy at 32 weeks postcontamination.
Mice fostered during the lactation period by a contaminated mot her accumulated respectively 0.9 cGy after 13 weeks and 1.5 cGy after 24 weeks due to americium-241 contamination of the milk. In the male mice contaminated 32 days before conception, the cumulative dose to the testis amounted to 2.1 cGy.

At 95 weeks LTC from contaminated mice showed a decreased CFU-GM output but no significant differences were seen between the control and radiocontaminated mice, in bone marrow cellularity and stem cell concentrations. The associated cumulative dose to the femur at the time was 8.5 cGy.
Preliminary results indicate that haemopoietic pluripotent stem cells (HPSC) can be maintained on a stromal layer.
Phenotypic characterization of the confluent adherent stromal layer yielded no obvious differences in cell types and extracellular matrix components between stromal layers derived from control animals and americium-241 contaminated mice.

The transplacental uptake of maternal plutonium-239 contamination was investigated and its effects on the development and maintenance of haemopoietic tissue were compared.
The repopulation of haemopoietic and stromal precursor cell populations 6 months after gamma-irradiation were compared at age 1 week, 4 weeks and 11 weeks.

Approximately 1% of plutonium-239 used to contaminate pregnant mice at mid term gestation appears in the newborn offspring, mostly in the foetal liver. When contamination occurs early in gestation, uptake in the newborn offspring is 10-fold lower. Effects on haemopoiesis in the long term however, are comparable. Reduced numbers of stem cells are required to maintain a higher level of proliferation in order to maintain cell output. The mechanisms in the 2 situations are different: one is determined primarily by effects on the haemopoietic stem cells; the other by effects primarily on the stromal microenvironment.

In control animals, the number of marrow cells per femur increased from about 1E7 at 4 weeks of age to 1.8E7 at 37 weeks. The corresponding increases for day 8 colony forming unit in spleen (CFU-S) were from 3000 to 4000, for day 12 CFU-S 3000 to 5000, and for iv-CFC 30000 to 55000. In contrast, CFU-F numbers declined from around 750 at 4 weeks of age to 400 at 15 weeks, and then increased to 650 at 37 weeks.These are greater increases in these parameters with time for younger mice compared with adults. If growth is reduced by irradiation, this indicates the greater potential for greater residual injury in the younger animals.

Regarding CFU-S response and recovery after external irradiation: there was no significant dose rate effect between 6 and 60 cGy/min for acute survival, acute survival levels were consistently higher for day 12 than for day 8 CFU-S, but the difference disappeared by 6 months postirradiation recovery and long term recovery at 6 months was equally good in mice of all ages, except perhaps after the highest dose (4.5 Gy) used in the 1-week old mice. Regarding CFU-S per colony, the recovery levels were in general lower in 1-week old mice than in the other ages, the effects of radiation on this endpoint were greater than for CFU-S numbers, in mice aged 1 week to 10-12 weeks, but not 4-5 weeks. There was a tendency for better recovery after lower doses delivered at the lower dose rate (6 cGy/min) in mice aged 1-week or 4 to 5 weeks, but this was not seen in adults. Surprisingly, recovery in adults was poorer after higher doses at the lower (compared to the higher) dose rate.

Concerning recovery of iv-CFC there was no significant dose rate effect and better recovery than CFU-S in 4-week old mice but with similar levels in 1-week old mice or in adults.
Regarding CFU-F an acute dose rate effect was observed for 4 to 5 week old mice but the reverse was found for adult mice. Recovery was in general poorer in 1-week old mice than in the other ages and a marked dose rate effect was observed for the levels of recovery in 4-week old mice. This was not so marked for adult mice, and there was a tendency towards the reverse effect in 1-week old mice.

The analysis and purification of stem cells from mouse bone marrow and from fetal liver were carried out in order to estimate their long term culture capability on irradiated stromal layers.
In a first series of experiments it was established that pluripotent haemopoietic stem cells could be highly purified from adult mouse bone marrow at the TNO institute in Rijswijk, and that those cells could be cultured SCK/CEN laboratory on a variety of stromal layers to produce haemopoietic progenitor cells. In a preliminary series of experiments the enrichment for the cell producing colony forming unit in granulocytes and macrophages (CFU-GM) on stroma was found to be a factor of 1.8. Further experiments are necessary to improve the isolation procedure in a combination of sorting techniques, and to compare different sorted cell fractions and control groups on the stromal layers.

The first experiments to isolate stromal progenitor cells in Rijswijk were not successful in that the sorted cells did not rise to stromal layers after transport to Mol. Unfractionated cells sent from Rijswijk to Mol gave normal stromal layers. Attempts to obtain stromal layers from sorted mouse cells failed, whereas in Rijkswijk good layers could be obtained from monkey bone marrow using fluorescence activated cell sorting of antibody labelled cells. Not only did mouse cells that were passed through the fluorescence activated cell sorter, fail to procedure a stromal layer, but also those cells that were separated using immunomagnetic bead procedures. The bead selection procedure is now being modified in Mol to prevent transportation problems. As soon as the reason for this unexpected result has become clear, further attempts will be undertaken to isolate the murine stromal stem cells by fluorescence activated cell sorting.

Research was carried out in order to determine whether residual damage is manifest by functional disorder in mature granulocytes (phagocytosis dysfunction) after 5 Gy whole body X-irradiation. The study focused on superoxide anion production in granulocytes obtained from peripheral blood and long term bone marrow culture (LTBMC).

The long term effect of radiation were the same for granulocytes obtained from peripheral blood or LTBMC; an enhancement of superoxide anion production in cells from irradiated animals. The difference was statistically significant at both 6 months and 12 months postirradiation. The number of nonadherent cells in the supernatant was always less in LTBMC from treated animals caused probably in these mice because the adherent layer never became properly established over the period studied.
The cell free supernatant obtained from LTBMC prepared from irradiated animals was able to stimulate granulocyte and macrophage colony forming cells (GM-CFC), showing that some colony stimulating factor (CSF) was produced in these cultures which was not detectable in cultures of normal marrow cells.
The data suggest that total body irradiation (5 Gy) of mice produce a compensatory mechanism probably in stromal cells, so that they release excess CSF. This factor could prime granulocytes in such a way that their capacity to produce superoxide anion is increased.

Research was carried out in order to compare the long term haemopoietic damage induced by a single acute dose of 7 Gy X-rays in adult and suckling mice and to achieve an efficient haemopoietic stem cell infection with retroviruses that allow the characterization of haemopoietic stem cell clones.

The long term study of haemopoiesis in mice showed that fewer than 10% of the animals died of irradiation syndrome; those which recovered showed essentially normal leukocyte numbers and haematocrit values in the first year postirradiation. However, a significant decrease in numbers of leukocytes was observed at later times. In the bone marrow, however, all the haemopoietic progenitors tested were significantly reduced to about 10 to 40% of age matched control values with no signs of recovery up to the end of the first year postirradiation. No evidence was found of a compensatory enhancement of haemopoiesis in the spleen where there was also a 10 to 40% depletion of haemopoietic progenitors compared with age matched controls.

Survival in suckling mice was comparable with that seen in adults, but in contrast to the results obtained in adults, a slow but consistent recovery of femoral haemopoietic progenitors was seen throughout the first year postirradiation. With the exception of colony forming unit in granulocytes and macrophages (CFU-GM) all haemopoieticprogenitors reached normal values in this period. In the spleen there was significant haemopoietic injury although less than that seen in adults.

From experiments on the transfer of genetic markers in haempoietic stem cells it was concluded that the infection of the multipotent haemopoietic precursor has been efficiently achieved. The aggressive selection step in the neomycin analogue, although efficient to increase the percentage of genetically marked colony forming unit in the spleen (CFU-S) can be omitted when retroviral titres were increased. Preliminary polymerase chain reaction (PCR) experiments of bone marrow obtained from recipients reconstituted with infected cells, showed a high expression of marked stem cells 8 weeks after transplantation, when preincubation was carried out in interleukin-3 (IL-3) plus interleukin-6 (IL-6).
At present, insertional analyses by Southern blotting of blood and lymphohaemopoietic organs from recipients transplanted with genetically marked bone marrow are in progress.

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CSC - Cost-sharing contracts

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MRC Radiobiology Unit
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Chilton
OX11 0RD Didcot
United Kingdom

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Last update: 2 April 1991

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