Tumour induction by methyl-nitroso-urea following preconceptional paternal contamination with plutonium-239.

We have investigated the possibility that transgenerational effects from preconceptional paternal irradiation (PPI) may render offspring more vulnerable to secondary exposure to an unrelated carcinogen. 239Pu (0, 128 or 256 Bq g(-1)) was administered by intravenous injection to male mice, 12 weeks before mating with normal females. Two strains of mouse were used -- CBA/H and BDF1. Haemopoietic spleen colony-forming units (CFU-S) and fibroblastoid colony-forming units (CFU-F), a component of their regulatory microenvironment, were assayed independently in individual offspring at 6, 12 and 19 weeks of age. Bone marrow and spleen from each of these mice were grown in suspension culture for 2 or 7 days for assessment of chromosomal aberrations. Female BDF1 were injected with methyl-nitroso-urea (MNU) as a secondary carcinogen at 10 weeks of age and monitored for onset of leukaemia/lymphoma. Mean values of CFU-S and CFU-F were unaffected by preconceptional paternal plutonium-239 (PP-239Pu), although for CFU-F in particular there was an apparent increase in variation between individual animals. There was significant evidence of an increase in chromosomal aberrations with dose in bone marrow but not in spleen. By 250 days, 68% of MNU-treated control animals (no PPI) had developed thymic lymphoma (62%) or leukaemia (38%). The first case arose 89 days after MNU administration. In the groups with PPI, leukaemia/lymphoma developed from 28 days earlier, rising to 90% by 250 days. Leukaemia (65%) now predominated over lymphoma (35%). This second generation excess of leukaemia appears to be the result of PPI and may be related to inherited changes that affect the development of haemopoietic stem cells.

Induction of lympho-haemopoietic malignancy: impact of preconception paternal irradiation.

PURPOSE: To investigate the effects of preconception paternal irradiation (PPI) from injected 239Pu on the susceptibility to induction of lympho-haemopoietic malignancy by subsequent irradiation or exposure to a chemical carcinogen. MATERIALS AND METHODS: The male CBA/H and DBA2 mouse was injected with 0, 128 or 256 Bqg(-1) 239Pu 12 weeks before mating with the normal CBA/H and C57B1 female respectively. CBA/H offspring were exposed to 3.3 Gy gamma-rays total body irradiation: BDF1 offspring were injected with 50 mg kg(-1) methyl nitrosourea (MNU). The offspring were assayed for changes in bone marrow progenitor cell numbers and chromosome aberrations and were followed up for subsequent induction of neoplasia. RESULTS: While the untreated mouse showed a normal distribution for cellularity, spleen colony-forming units (CFU-S) and fibroblastoid colony-forming units (CFU-F), significant numbers of PPI offspring presented levels outside the normal range. There was a tendency for them also to show increased, dose-related, levels of chromosomal aberrations. Offspring treated with irradiation or MNU developed an increased incidence of lympho-haemopoietic malignancies. CONCLUSIONS: These studies have shown that PPI results in offspring that are more susceptible to the induction of lymphohaemopoietic malignancy on encountering a secondary carcinogenic insult. This may be linked to inherited chromosomal instability and abnormal kinetics of haemopoiesis. The experiments indicate a potential mechanism by which an increased incidence of leukaemia may be linked to PPI.

Myeloid leukaemia and osteosarcoma in CBA/H mice given 224Ra.

Four groups of 400 12-week-old CBA/H mice were injected i.p. with 69, 139, 280 and 550 Bqg-1 224Ra. A further group of 400 mice were injected i.p. with diluting solution only. The mice were then allowed unrestricted access to food and water until they died or were killed. 53 cases of myeloid leukaemia and 22 cases of osteosarcoma were confirmed in the 2000 mice injected, and for both tumour types direct relationships were shown to exist between the amount of 224Ra administered and the incidence of tumours. It is concluded that mouse is at a greater risk from myeloid leukaemia than from osteosarcoma in the region of administered 224Ra below that which causes a maximum yield of osteosarcoma. These results are discussed in the light of the present acceptance of osteosarcoma as the major risk to man from bone-seeking alpha-particle emitters.

Long-term effects of plutonium-239 and radium-224 on the distribution and performance of pluripotent haemopoietic progenitor cells and their regulatory microenvironment.

Experiments are described which investigate the long-term damage to haemopoietic progenitor cells (CFU-S) and their microenvironment in mouse marrow resulting from the administration of leukaemogenic amounts of plutonium-239 and radium-224. 239Pu (35 Bq g-1 body weight) and 224Ra (555 Bg g-1 body weight) were injected into 10-12-week-old mice, and numbers, proliferative activity and self-renewal capacity of CFU-S were measured at different locations in femoral marrow at intervals over the following 2 years. Parallel measurements were also made of the quality of the haemopoietic microenvironment by ectopic transplantation of bone marrow cells. There was some recovery from the initial effects of 239Pu on CFU-S numbers after 3-6 months, although the recovery was not maintained in all marrow fractions. Following 224Ra administration there was an initial transient increase in CFU-S numbers in the fraction of marrow furthest from bone surfaces but a considerable depression in numbers in other regions of marrow; there was no recovery between 3 and 6 months and subsequent recovery was not complete in all regions of marrow. The differential responses of CFU-S and the haemopoietic microenvironment following 224Ra or 239Pu administration seemed in some ways related to the metabolism of the radionuclides. There was a profound reduction in the ability of marrow to generate ossicles when transplanted under the kidney capsule as a result of the administration of either 224Ra or 239Pu, with only transient recoveries from the effects of 239Pu at 4 days and at 3 months after injection.

On the late seeding of CFU-S to the spleen: 8- vs 12-day CFU-S.

Marrow from 5-fluorouracil- or cyclophosphamide-treated mice, injected into lethally irradiated recipients, gives an increasing number of spleen colonies between days 7 and 14. It has been suggested that the later-forming colonies result from the more primitive spleen colony-forming units (CFU-S), which first seed into the marrow, only later to be recirculated and form colonies in the spleen. Strontium 89 (89Sr), a bone-seeking radionuclide, was injected into recipient mice to block such putative recirculation. A dose of 89Sr, which killed at least 99.8% of CFU-S in, or entering, the bone cavities, was incapable of preventing the increase in spleen colony numbers. Similarly, the splenic environment, modified by the presence of spleen colonies and able to provide a better bed for trapping CFU-S from the peripheral circulation, yielded the same number of further CFU-S, whether or not the animal had received 89Sr. Thus, it was concluded that the 12-day CFU-S does not seed initially into the marrow spaces. Direct observation of the quality of CFU-S initially seeding into the bone marrow and spleen showed, by retransplantation into secondary irradiated mice, that a similar spectrum of CFU-S types had seeded both organs.

The induction by 239Pu of myeloid leukaemia and osteosarcoma in female CBA mice.

Plutonium-239 was injected into 12-week-old female CBA/H mice in the range 1.85-18.5 kBq kg-1 either as a single injection or as 16 injections spaced at 3.5 day intervals over eight weeks. There was a highly significant increase in the yield of fully developed osteosarcomas with increased amounts of 239Pu for both modes of injection. Osteosarcomas too small to be diagnosed radiographically were also seen in many bones and small but significant yields of myeloid leukaemia were seen in animals given plutonium. Although more myeloid leukaemia was seen in the mice given plutonium in divided amounts than in those given the plutonium in a single injection it could not be shown that multiple injection significantly affected the yield of either late effect.

Effects of plutonium-239 on haemopoiesis. I. Quantitative and qualitative changes in CFU-S in different regions of the mouse femur and vertebrae.

Mice were injected with plutonium-239 (960 Bq/mouse) and, over a period of four months, the response of haemopoietic tissue and the self-renewal capacity of its stem cells was monitored. Cellularity, CFU-S concentration and self-renewal capacity were measured in five different regions of bone and marrow--axial and marginal marrow of the femoral shaft, femur shaft, proximal and distal ends of the femur shaft and vertebrae. Cellularities were little affected by plutonium but CFU-S were reduced in all regions, most severely in the bone shaft and marginal marrow due to the initial deposition of plutonium on the bone surface, by four days. The reduction in axial CFU-S, however, was due probably to a relatively long plasma half-life resulting from the tendency of plutonium to combine with plasma proteins. The capacity of CFU-S for self-renewal was reduced and remained low in all zones. Thus, although the highly self-renewing axial CFU-S were depleted, and remained so, due probably to a longer term redistribution of plutonium throughout the marrow, additional proliferation of the more mature CFU-S in the other zones kept their self-renewal low while replenishing their numbers and maintaining a normal cell output.

The induction by 224Ra of myeloid leukaemia and osteosarcoma in male CBA mice.

Radium-224 was injected into 12-week-old male CBA mice in the range 2-64 kBq per mouse either as a single injection or as eight injections spaced at 3.5 day intervals over 4 weeks. Small but significant yields of myeloid leukaemia or osteosarcoma were obtained in all but the control groups. An effect of mode of administration (single or multiple injections) could not be demonstrated but the combined results showed: a maximum yield of myeloid leukaemia in the region 8-16 kBq 224Ra; a greater yield of osteosarcoma than myeloid leukaemia at 64 kBq 224 Ra injected.

Age-related and 224Ra-induced abnormalities in the teeth of male mice.

A high incidence of incisor abnormalities was found in aged control and aged 224Ra-treated male CBA mice. Visual examination of the abnormalities in both controls and treated mice revealed extreme shortening of the upper incisors and hypoplastic, grooved or undulating enamel. The administration of 865 or 1730 nCi of 224Ra hastened the onset of incisor abnormalities although no specific feature was attributable solely to radium toxicity. Radiography and histology revealed corrugated incisors, obliteration of the pulp cavity, extension and disorganized growth of incisors basally, secondary incisors, open pulp and fractures within the alveoli. There was a statistically-significant reduction in the number of molars present in animals given 432, 865 or 1730 nCi 224Ra.

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.