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.     

The absorption of ingested neptunium, plutonium and americium in newborn hamsters

Hamsters aged 1, 4, 7, 22 and 30 days were given oral doses of either plutonium-239 citrate or americium-241 nitrate. The values of gastrointestinal absorption obtained were 3.5, 1.4, 0.04, 0.007 and 0.003 per cent, respectively, for plutonium and 4.5, 1.7, 0.5, 0.006 and 0.02 per cent, respectively, for americium, compared with values in adults of 0.01 per cent for plutonium and 0.05 per cent for americium. The absorption of neptunium was measured in hamsters aged 2 and 4 days and values of 2.3 and 1.7 per cent, respectively, were obtained for 239Np as the nitrate and 5.5 and 2.1 per cent, respectively, for 239Np as the bicarbonate compared with the values in adults of 0.02 per cent for both chemical forms. Thus, the absorption of plutonium, americium and neptunium at 1-2 days of age was about 100 times greater than in adults. The results for plutonium and americium show that absorption decreased rapidly with age over the suckling period. The values of absorption obtained at the time of weaning at 22 days were lower than in adults.     

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.     

Reduced weight in mice offspring after in utero exposure to 2450-MHz (CW) microwaves

Time-bred CD-1 mice (100) were sham-irradiated or irradiated with 2450-MHz (CW) microwaves at 28 mW/cm² for 100 minutes daily from the 6th through 17th day of gestation. The offspring were examined either as fetuses after hysterotomy on the 18th day of gestation or as naturally born neonates on the 1st and 7th day of age. Fetuses of half of the dams were examined on the 18th day of gestation. The incidence of pregnancy and the numbers of live, dead, resorbed, and total fetuses were similar in both groups. The mean weight was significantly lower (10%) in live microwave-irradiated fetuses, and ossification of sternal centers was significantly delayed. In the offspring that were born naturally, the mean weight of microwave-irradiated 7-day-old suckling mice was significantly lower (10%) than that of the sham-irradiated group. Survival rates of neonates in these two groups were not different. These data demonstrate that the decreased fetal weight seen in microwave-irradiated mice is retained at least 7 days after birth. Evidence from other published studies is presented to show that the retarded growth is persistent and might be interpreted as permanent stunting.     

The induction of liver tumors by 239Pu citrate or 239PuO2 particles in the Chinese hamster

The influence of radiation dose distribution on the frequency of 239Pu-induced liver tumors was evaluated in the Chinese hamster. Different concentrations of 239Pu citrate 239PuO2 particles of known sizes were injected intravenously via the jugular vein. About 60% of the injected 239Pu citrate was deposited in the liver and 40% in the bone. The 239Pu citrate was rather uniformly distributed throughout the liver parenchyma. Injected plutonium oxide particles were taken up by the reticuloendothelial system with 90% of the body burden deposited in the liver. The 239PuO2 particles were localized in the Kupffer cells and produced nonuniform dose distributions that were dependent on particle size. There was an activity- and dose-dependent increase in the incidence of total liver parenchymal cell tumors following injection with either plutonium particles or citrate. For animals that received 14.0-, 2.7-, 0.3-, and 0.04-Gy dose to liver from 239Pu citrate the cumulative tumor incidence was 39, 32, 5, and 0%, respectively. Animals that were injected with the 0.24 micron 239PuO2 particles had doses of 42.0, 7.2, and 0.8 Gy to the liver and tumor incidences of 34, 26, and 5%, respectively. Plutonium citrate also produced hemangiosarcomas of the liver and tumors in bone and bone marrow. The latent period for liver tumor appearance in animals exposed to 239Pu citrate or 239PuO2 particles increased as the injected activity decreased. For animals injected with a similar total activity (7.4 Bq/g), the lifetime cumulative liver tumor incidence was similar for animals exposed to either 239Pu citrate (32%) or 239PuO2 (26%). There was little effect of particle size on liver tumor incidence. These data indicate that, in Chinese hamster liver, local radiation dose distribution is less important in altering tumor incidence than injected activity or average dose. However, the more uniform irradiation from 239Pu citrate administration was more effective in cancer production than the nonuniform irradiation from 239PuO2 particles.     

Effect of plutonium-239 on the mitotic activity of mouse bone marrow cells

Summary Mitotic index of the bone marrow cells was studied in femoral bone marrow of mice given 313 kBq²³⁹Pu kg^–1. The attention was turned to the femoral midshaft and the mitose concentration, intensified by Colcemid stathmokinetic effect, was evaluated in a sampling field from endosteal surface to the central venous canal, throughout 68 weeks. It has been found that the plutonium effect in the sampling band is rather uniform except the points in subendosteal zone early after plutonium injection, where the mitotic index was reduced in such a way that the mitotic gradient, observed in controls, was affected. The mitotic activity in femoral diaphysis of plutonium injected mice was mobilized approximately till the 30th week of contamination. Later it deteriorated progressively. The results are discussed and should not be regarded as representative for the entire bone marrow hemopoiesis.     

The gastrointestinal absorption of organically bound forms of plutonium in fed and fasted hamsters

Values of about 0.005-0.01 per cent were obtained for the absorption in fed hamsters of plutonium ingested as Pu4+ citrate, isocitrate, phytate and malate complexes and Pu3+ ascorbate compared with about 0.003-0.004 per cent for Pu4+ nitrate. Replacing drinking water with tea did not affect the result for Pu4+ nitrate. Fasting hamsters for 8 h before the administration of plutonium citrate increased absorption to 0.1-0.2 per cent. An extra period of fasting for 4 h after administration did not lead to a further increase in absorption. Similar values were also obtained when plutonium citrate was administered after a 24 h fast, followed either by immediate access to food or a further 4 h fast. In hamsters fasted for 24 h before administration of either Pu3+ ascorbate or Pu4+ nitrate, about 6-7 per cent of the ingested plutonium was retained in the gastrointestinal tract after one week. At three weeks after ingestion of Pu3+ ascorbate, gastrointestinal retention had fallen 100-fold without an increase in absorption.     

The distribution of plutonium-214 in rodents.

Plutonium-214 citrate solution at pH 6-5 was injected intravenously or intra-peritoneally into hamsters and rats at a dose of 50 MBq kg-1 (1-35 mCi kg-1). The animals were killed 1 day or 1 week later, and tissues were removed for autoradiography and radiochemical analysis. Plutonium-241 was distributed in rats in the same way as plutonium-239, and is a suitable isotope for high-resolution tissue-section autoradiography. Plutonium deposits in cells consisted of a nuclear and a cytoplasmic component. In the hamster kidney cells, the amount associated with the nucleus was about 55 per cent of the total cellular plutonium at 24 hours after injection. Six days later, it was only about 30 per cent. Plutonium deposits were also characterized in hepatocytes, in the interstitial cells of the testes, in the cells of ovarian follicles, in chondrocytes and in bone cells, including osteoblasts and osteocytes. In bone there appeared to be both an extracellular and intracellular deposit. No evidence was found of substantial incorporation of plutonium into the mineral phase of bone.     

Effects of duration of fast and animal age on the gastrointestinal absorption of plutonium

The fraction of plutonium absorbed after oral administration of Pu(VI) to 24-h-fasted mice was 19 X 10(-4), 13-fold higher than in fed mice, 1.4 X 10(-4). We have investigated the relevance of the high gastrointestinal (GI) absorption value for the 24-h-fasted animals in setting drinking water standards for humans. When fasting was initiated at the beginning of the active phase of the mouse's daily activity cycle (when they would normally eat), plutonium GI absorption rose from 2.8 X 10(-4) at zero-time to a level typical of the 24-h-fasted mouse after only 2 h of fasting. In contrast, in mice allowed to eat for 4 h into their active phase prior to initiation of the fast (meal-fed mice), 8 h of fasting were required before GI absorption rose to a level similar to that of the 24-h-fasted mouse. The fraction of plutonium retained after gavage administration of Pu(VI) to 1-day-old rats was 74 X 10(-4), 70-fold higher than the value for fed adults. Retention after GI absorption in neonates remained 30- to 70-fold higher than in adults until weaning. One week after weaning, the fraction absorbed and retained by fed weanling rats was the same as that for fed adults, 1 X 10(-4). Drinking water standards for plutonium have been set based on GI absorption values for fed adult animals. The 10- to 100-fold increases in plutonium absorption in young and fasted animals reported by ourselves and others, and the rapid rise to fasted levels of absorption at the start of the animal's active phase, indicate that consideration should be given to elevated levels of plutonium absorption in young and fasted individuals.     

Uptake and clearance of plutonium-238 from liver cells transplanted into fat pads of F344 rats

This research is directed toward understanding the role of liver cells and the liver environment in plutonium biokinetics. Animals injected with liver cells and control animals received a single intraperitoneal injection of 37 kBq (1 microCi) 238Pu citrate and were serially sacrificed 1, 5, 10, 15, 30, 60 or 70 days later. Uptake, retention and distribution of Pu in intact liver and in liver cells growing in fat pads were determined. From these measurements, it was observed that the cells of the intact liver took up about twice as much 238Pu as liver cells transplanted into the fat pads of the same animal. The retention half-life was 8.3 days for the total activity in the liver, 20 days using tracks/cell measurements in the liver and 16 days for the tracks/cell measurements in the liver cells translocated to fat pads. When the data on tracks/cell were standardized relative to the amount of Pu present at 5 days after the injection, there was no significant difference between the retention of Pu in liver cells from intact animals and liver cells transplanted into the fat pads. About 20 per cent of the 5-day Pu liver burden in both liver cells and liver cells transplanted into fat pads was retained at 70 days. The smaller retention and clearance for liver cells in different environments indicate that uptake and clearance of Pu from the body is dependent, to a major extent, upon hepatocyte function.     

Quantitative microscopic studies of the distribution and retention of 239Pu in the ilium of the female CBA mouse.

Three month old female CBA mice were injected with 50 nCi kg-1 body mass of minimally polymeric 239Pu-citrate and killed at 24 hours, 10 days, 1 month and 3 months after injection. The distribution of 239Pu in the ilia of these mice was analysed using neutron-induced autoradiography of bone sections together with computer-based methods of data reduction. Results of these investigations demonstrate that while 239Pu is initially localized on bone surfaces, by 3 months after injection it is fairly uniformly distributed throughout mineral bone and its included marrow.     

Combined prophylactic administration of riboxin and algisorbum at 239Pu intake into gastrointestinal tract of rats

The present study is devoted to the investigation of effectiveness of combined prophylactic administration of riboxin and algisorbum at 239Pu per oral intake and possible mechanisms of their interaction, and also to comparative estimation of effectiveness of combined administration of the preparations at per oral and intra peritoneal methods of riboxin introduction. The experiments have been carried out on white nonlinear rats. Riboxin (per oral and intra peritoneal) and algisorbum (per oral) have been introduced to the rats both separately and combined before per oral 239Pu introduction. Data obtained as a result of the investigation showed that combined riboxin and algisorbum introduction into gastrointestinal tract before 239PU intake lead to greater decrease in the plutonium content in the organs of deposition than single algisorbum administration. Intra peritoneal riboxin introduction reduced effectiveness of per oral algisorbum administration in plutonium binding in GI tract. Efficiency of combined riboxin and algisorbum administration in the reduction of 239Pu accumulation in organs depends on the method of riboxin introduction and develops only at per oral introduction.     

Distribution of 239Pu in the skeleton of the tree shrew (Tupaia belangeri) between 15 and 50 months after injection

The macroscopic and microscopic distribution of intramuscularly injected, essentially monomeric, 239Pu was studied in the skeleton of the adult tree shrew (Tupaia belangeri). Data for the period between 15 and 50 months after injection are presented and compared with the data from earlier time points. Between 83 and 500 days after injection the nuclide content and the wet weight of the skeleton decreased to a constant level at about 55 per cent of the maximum values. The microscopic distribution has been analysed in distal femora, proximal humerus, proximal tibia and lumbar vertebra over the whole observation time; additionally at some selected time points proximal femur, femur shaft, distal humerus and distal tibia were analysed. The initial endosteal surface activity ranged from 3.8 to 5.3 Bq/cm2 and decreased to a minimum at about 1000 days after injection and increased thereafter. A similar behaviour was found for the dose rate near bone surfaces which was initially about 0.075 Gy/day on endosteal surfaces. In the deep bone and the deep marrow the dose rate was negligible, about 0.008 Gy/day and 0.001 Gy/day, respectively. The average cumulative dose 1500 days after injection was about 67 Gy on the endosteum, six times greater than the cumulative dose calculated from the mean concentration of plutonium in the whole skeleton. All values are normalized to an injected activity of 37 kBq/kg body weight. The tupaia data are discussed in relation to the available data from monkeys, dogs and rats.     

Association of plutonium with lysosomal, lipofuscin-like granules in Chinese hamster hepatocytes: evidence from electron microscopic and biochemical studies with 241Pu and 239Pu

This electron-microscopic-autoradiographic study was undertaken to identify the cell organelles, which bind plutonium in Chinese hamster hepatocytes at different times after injection. Female Chinese hamsters were injected intraperitoneally with 241Pu and sacrificed at time intervals of between 4 days and 35 weeks. The Chinese hamster was chosen as the experimental animal as it is a species in which there is virtually no elimination of plutonium from the liver. From the 4th day onwards beta-tracks were found over globular electron-dense structures, which were randomly distributed in the cytoplasm of the hepatocytes and strongly resembled lipofuscin bodies. Comparison of the results with those from biochemical experiments showed good agreement between the morphological and biochemical observations. At early times after injection 241Pu was also found in the hepatocyte nuclei. All the evidence suggests that in this species plutonium in hepatocytes becomes bound to lipofuscin-accumulating lysosomes, which cannot be excreted.     

Reciprocal translocations in ageing mice and in mice with long-term low-level 239Pu contamination

Single intravenous injections of 185 Bq monomeric 239Pu were given to male mice, and the frequency of primary spermatocytes with reciprocal translocations, determined 724 days after treatment, was not significantly different from that of age-matched untreated controls. These old animals showed significantly higher aberration frequencies than young adults. The data therefore show that for low initial activity and very long retention time the possible cytogenetic effects of incorporated nuclide does not change the age-related pattern of increase of spontaneous chromosome aberrations. Considerations of the main variables involved in the induction of cytogenetic effects of incorporated plutonium, based on literature data, indicate that the initial injected activity, the estimated total accumulated average organ dose, and the retention time interact in a complex way; as far as can be seen at present, the effects seem to be dependent mainly on the initial activity at short times after contamination, while the retention time appears to be predominant in the case of long-term observations.     

Removal of Pu and am from beagles and mice by 3,4,3-LICAM(C) or 3,4,3-LICAM(S)

Decorporation of Pu and Am by tetrameric catechoylamide (CAM) ligands has been investigated in beagles and mice. Eight dogs were injected intravenously (iv) with 237 + 239Pu(IV) + 241Am(III) citrate, and 30 min later, pairs of dogs were injected iv with 30 mumole/kg of 3,4,3-LICAM(C) [N1,N5,N10,N14-tetrakis(2,3-dihydroxy-5-sulfobenzoyl)tetr aazatetradecane, tetrasodium salt], 3,4,3-LICAM(S) [N1,N5,N10,N14-tetrakis(2,3-dihydroxy-4-carboxybenzoyl)te traazatetradecane, tetrasodium salt], CaNa3-DTPA, or each of the latter two ligands. Blood was sampled, and excreta were collected for 7 days, at which time the dogs were sacrificed and nuclide retention in liver and nonliver tissue was measured. Groups of five mice were each given 238Pu(IV) or 241Am(III) citrate iv; 3 min later 30 mumole/kg of a CAM ligand was injected intraperitoneally, mice were killed at 24 hr, and separated excreta and tissues were analyzed. In the dogs, average retention at 7 days of the injected Pu and Am, respectively, was as follows: 12 and 70% after treatment with a CAM ligand alone; 30 and 20% after DTPA; 12 and 20% after LICAM(S) plus DTPA; 90 and 89% without a ligand. In the mice, mean retention of the injected Pu and Am, respectively, was as follows: 14 and 66% after treatment with LICAM(C); 21 and 54% after LICAM(S); 91 and 87% without a ligand. In both species, about 99% of net Pu excretion (excretion with ligand - excretion without ligand) promoted in 24 hr by DTPA or LICAM(S) was in the urine, whereas about 10% of net Pu excretion promoted by the less hydrophilic LICAM(C) was in feces. Delayed excretion of both Am and Pu was significant in all ligand-treated dogs. Comparison of the nuclide content of tissues of ligand-treated mice with those of mice killed 3 min after nuclide injection indicated that the CAM ligands chelated circulating Pu and Am and prevented further deposition. In addition, the CAM ligands removed much of the presumably loosely bound Pu present in liver and skeleton at the time of ligand injection. LICAM(C) was more effective in removing Pu from liver and LICAM(S) was more effective in the skeleton. Moderate to severe uremia and histological evidence of cell killing in the distal tubules of the kidney were observed in the four dogs injected once with 30 mumole/kg of LICAM(S).(ABSTRACT TRUNCATED AT 400 WORDS)     

Decreased weight, DNA, RNA and protein content of the brain after neutron irradiation of the 18-day mouse embryo

Pregnant mice were irradiated with 0.5 Gy fission neutrons on the eighteenth day of their gestation. The average litter size at birth was unchanged but mortality increased 5-6 fold in the first 3 days. The irradiated mice were the same weight as control mice at birth but showed a progressively increasing weight deficiency up to at least 36 days as compared to controls. Brain weight was 37, 45 and 25 per cent less in 2-, 3- and 52-week old irradiated animals, respectively, and the ratio of brain weight to body weight was 25, 27 and 13 per cent less. The concentrations of DNA, RNA and protein (mg/g wet tissue) were the same in irradiated and control mice in both brain and liver at all three ages. Total DNA, RNA and protein contents of whole brain after irradiation were 56-75 per cent of the control levels. No definite decrease was observed in liver. Histological study at 6 hours after irradiation showed nuclear pyknosis in the central nervous system from definite to very severe according to the part examined. It is concluded that damage to the central nervous system of the 18-day mouse foetus after neutron irradiation is mainly due to killing and/or inhibition of the differentiation of neuroblasts.     

The effect of X rays, DTPA, and aspirin on the absorption of plutonium from the gastrointestinal tract of rats

To measure the effect of radiation on plutonium transport, rats that were exposed to 250-kVp X rays were given 238Pu 3 days afterwards by either gavage or injection into a ligated segment of the duodenum. In a second group of experiments, rats were either injected intraduodenally with 238Pu-DTPA or administered the chelate intravenously and the 238Pu by gavage. In a third experiment, rats that had been gavaged with 200 or 400 mg/kg/day of aspirin for 2 days were injected intragastrically with 238Pu nitrate. Results of the first experiment showed a dose-dependent increase in 238Pu absorption between 800 and 1500 rad of lower-body X irradiation. Intravenous or intraduodenal injections of DTPA caused a marked increase in 238Pu absorption but resulted in decreased plutonium deposition in the skeleton and liver. Retention of 238Pu in the skeleton of rats given aspirin was double that of controls, but the effect on plutonium absorption was less marked than that of DTPA.     

EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE RAT BRAIN

Abstract— Rats were undernourished by approximately halving the normal food given from the 6th day of gestation throughout lactation. Growth of the foetuses was nearly normal, in marked contrast to the severe retardation caused by undernutrition during the suckling period. In comparison with controls the size and the DNA content of the brain were permanently reduced by undernutrition during the suckling period: this effect was relatively small, approx. 15 per cent decrease at 21 and 35 days. The rate of ¹⁴C incorporation into brain DNA at 30 min after administration of [2-¹⁴C] thymidine was taken as an index of mitotic activity; compared with controls there was severe reduction in mitotic activity (maximal decrease by about 80 per cent at 6 days in the cerebrum and by 70 per cent at 10 days in the cerebellum). The rate of acquisition of cells was calculated from the slopes of the logistic curves fitted to the estimated DNA contents. In normal animals the maximal slope was attained at 2·7 days and at 12·8 days after birth in cerebrum and cerebellum respectively; the daily acquisition of cells at these times was 4·8 × 10⁶ and 18 × 10⁶ cells respectively. The fractional increase in cell number at the maximum was 5·4 percent per day in the cerebrum and 15·2 per cent per day in the cerebellum. The rate of acquisition of cells relative to the rate of mitotic activity was higher in the brains of undernourished animals than in controls. One of the compensatory mechanisms for the severe depression of mitotic activity in the brain of undernourished animals Seems to involve a reduction in the normal rate of cell loss.     

Activation of alveolar macrophages after plutonium oxide inhalation in rats: involvement in the early inflammatory response

Alveolar macrophages play an important role in the distribution, clearance and inflammatory reactions after particle inhalation, which may influence long-term events such as fibrosis and tumorigenesis. The objectives of the present study were to investigate the early inflammatory events after plutonium oxide inhalation in rats and involvement of alveolar macrophages. Lung changes were studied from 3 days to 3 months after inhalation of PuO2 of different isotopic compositions (70% or 97% 239Pu) and initial lung deposits (range 2.1 to 43.4 kBq/rat). Analyses of bronchoalveolar lavages showed early increases in the numbers of granulocytes, lymphocytes and multinucleated macrophages. The activation of macrophages was evaluated ex vivo by measurement of inflammatory mediator levels in culture supernatants. TNF-alpha and chemokine MCP-1, MIP-2 and CINC-1 production was elevated from 7 days after inhalation and remained so up to 3 months. In contrast, IL-1beta, IL-6 and IL-10 production was unchanged. At 6 weeks, pulmonary macrophage numbers and activation state were increased as observed from an immunohistochemistry study of lung sections with anti-ED1. Similarly, histological analyses of lung sections also showed evidence of inflammatory responses. In conclusion, our results indicate early inflammatory changes in the lungs of PuO2-contaminated animals and the involvement of macrophages in this process. A dose-effect relationship was observed between the amount of radionuclide inhaled or retained at the time of analysis and inflammatory mediator production by alveolar macrophages 14 days after exposure. For similar initial lung deposits, the inflammatory manifestation appears higher for 97% 239Pu than for 70% 239Pu.