Distribution and biological effects of inhaled 239Pu(NO3)4 in cynomolgus monkeys.

Twenty male cynomolgus monkeys were exposed by inhalation either to an aerosol of 239Pu(NO3)4 to produce projected initial lung burdens of either 40, 10, or 4 kBq or to a carrier aerosol as a control. Animals died or were sacrificed at 0.01, 1, 3, 6, 12, 24, 40, and 99 months after inhalation, and the distribution and biological effects of the 239Pu were determined. The 239Pu cleared efficiently from the lungs so that less than 0.05 kBq remained at 99 months after exposure to 40 kBq. Total skeletal 239Pu activity was nearly constant after the first year, but the fraction of the body burden in skeleton at sacrifice increased with time up to 99 months because of clearance from other organs. Plutonium in the liver increased to a peak at 1 year and then decreased to about 10% of the peak value at 99 months. Plutonium in the testes was localized in the interstitial tissue with only 0.01 to 0.002% of the projected lung burden remaining in testes at 99 months after inhalation. Three animals exposed to 40 kBq of 239Pu died of radiation-related pulmonary pneumonitis and fibrosis. A primary papillary adenocarcinoma of the lung was identified in one animal exposed to 40 kBq initial lung burden and sacrificed 99 months after inhalation. The frequency of chromosome aberrations in blood lymphocytes was significantly elevated only in monkeys with projected deposits of 40 kBq of 239Pu. There was no change in aberration frequency in other exposure groups as a function of inhaled activity, time after exposure, or calculated total dose to the lungs. Only in monkeys that had marked radiation-induced pathological changes in the lung did the frequency of chromosome-type aberrations increase significantly, to a value about twice the control level. In cynomolgus monkeys, chromosome aberration frequency in blood lymphocytes is not a good indicator of radiation dose or damage from inhaled soluble plutonium.     

Cardiopulmonary function of dogs with plutonium-induced chronic lung injury

Beagle dogs had signs of restrictive lung disease 1 to 5 years after exposure by inhalation to 239PuO2 aerosols. The 239PuO2 aerosols were monodisperse with activity median aerodynamic diameters of 0.75, 1.5, or 3.0 microns. The plutonium particles produced protracted alpha irradiation of the lungs. Ten dogs had specific initial pulmonary burdens (IPB) of 330 to 4,100 kBq of 239PuO2/kg of body mass. The average onset time of clinical signs of lung injury was 3 years after exposure; the average time from the onset of signs until cardiorespiratory function evaluation was 5.5 years. A second group of 10 dogs had IPB of 110 to 2000 kBq of 239Pu/kg of body mass but no signs of lung injury. A third group of 10 dogs, not exposed to 239Pu, were matched for age and sex. Cardiopulmonary function tests were performed. Only the dogs in group I with signs of lung injury had a mild respiratory function disorder consisting of smaller lung volumes, reduced compliance, increased respiratory frequency and minute volume, and reduced carbon monoxide diffusing capacity. Cardiac function of all three groups was similar. These findings indicate that alpha irradiation of the lungs of man could produce restrictive lung disease at long times after initial exposure.     

Preliminary studies of the interaction between 239PuO2 and cigarette smoke in the mouse lung

Our current experiments were designed to show whether 12 months' exposure to cigarette smoke enhances the incidence of lung tumours in mice that had previously inhaled 239PuO2. These periods of smoke exposure are almost complete. After death their lungs will be cleared and any nodules found will be sectioned for histopathology. This paper reports the results of two preliminary experiments conducted earlier. The first study showed that mice could tolerate the proposed smoking regime for 3 months, with no sign of ill health in any animal throughout. The major difference found was a reduced growth rate in both smoke- and sham-exposed mice relative to that of cage controls. After 3 months of treatment, histopathology and morphometry of lung sections found only slight smoke-induced changes. These included a reduced proportion of alveolar space and an increased number of pulmonary alveolar macrophages (PAM) per unit area. Bronchopulmonary lavage showed that the PAM from smoke-exposed mice were larger than those from sham-exposed or control mice and that an increased proportion of cells were binucleate. All mice in the second study were initially exposed to 239PuO2, then subsequently divided into three treatment groups as above. Cigarette smoke exposure was shown to inhibit the removal of 239Pu from the lung whilst sham exposure had no effect. Smoke exposure also produced an increase and sham exposure a decrease in lung weights relative to those of cage controls. The latter was probably as a result of their lower growth rate. In our current experiments it is likely that the group receiving 239PuO2, then smoke, will receive a higher radiation dose to lung than those receiving 239PuO2 only. Any increased tumour incidence found will be considered in conjunction with this evidence.     

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.     

Pathogenetic process of lung tumors induced by inhalation exposures of rats to plutonium dioxide aerosols

Sequential examinations were done on the pulmonary cytokinetics and pulmonary lesions in rats after inhalation exposure to (239)PuO(2) aerosols to investigate the pathogenesis of lung tumors. Total cell yields of lavaged bronchoalveolar cells as well as the estimated numbers of pulmonary alveolar macrophages were significantly reduced from 1 to 3 months after exposure but recovered thereafter to the control levels. The proportions of multinucleated or micronucleated pulmonary alveolar macrophages increased significantly in lavaged cells from 1 month, and the increase was sustained up to 18 months after exposure. Both tumor necrosis factor and nitric oxide were shown to be differentially released from stimulated cultures of pulmonary alveolar macrophages during the period from 6 to 18 months after exposure. The labeling indices of alveolar and bronchiolar epithelial cells treated with 5-bromo-2'-deoxyuridine increased significantly in lungs from 3 months and were sustained up to 18 months after exposure. Histopathological examinations revealed that after the early inflammation, hyperplasia and metaplasia of the lining of the bronchioloalveolar epithelium were predominant from 3 to 6 months, while adenomatous or adenocarcinomatous lesions appeared and developed from 12 months after exposure. The appearance of primary lung tumors, almost all of which were adenomas and adenocarcinomas, was found in the dose range of 1 to 2 Gy from 12 months after exposures. These results indicate that the pathogenetic process initiated by early cellular damage and alterations associated with inflammation is followed by the proliferative and metaplastic lesions of pulmonary epithelium, leading to the appearance and development of pulmonary neoplasms from 1 year after the inhalation exposures in rats that received a minimum lung dose of more than 1 Gy.     

The subcellular distribution of 238Pu and 239Pu in primary cultures of rat hepatocytes

The subcellular distribution of 238Pu and 239Pu after incubation of primary cultures of rat hepatocytes with the citrate complex of these metals was studied, and the results were compared with data from in vivo experiments. As in vivo, the lysosomes are the principal organelles in which 238Pu and 239Pu are accumulated. In contrast to in vivo studies, 239Pu is also detectable on the pericellular membranes and in the cell nuclei, where it is predominantly bound to a high-molecular-weight component. The percentage of the total cellular 239Pu which can be recovered in the cell nuclei increased with incubation time from 10% at 1 h to nearly 30% at 5 h. Plutonium-238, an isotope with 270-fold higher specific activity than 239Pu, showed no association with the nuclei. The membrane-bound fraction of 239Pu, as determined using the exogenous chelator diethylenetriaminepentaacetic acid decreased from 30% at shorter incubation times to 15% at longer incubation periods. After incubation with 238Pu the membrane fraction and the cytosolic fraction contained higher concentrations of the radionuclide than after incubation with 239Pu.     

The ultrastructure of mouse testicular interstitial tissue containing plutonium-239 and its significance in explaining the observed distribution of plutonium in the testis.

The technique of autoradiography with Araldite-embedded sections was used to study the distribution of 239Pu in mouse testis at various times post-injection. Adjacent sections were examined with both the light microscope and electron microscope. The autoradiographs showed that from 1 week to 3 months postinjection, most 239Pu in located in interstitial tissue. The major change in distribution observed was that the early diffuse deposit in interstitial tissue is concentrated in macrophages with increasing time post-injection. This is a real change of distribution as the amount of 239Pu in mouse testis remains constant from 1 week to 3 months post-injection. Study of the ultrastructure of interstitial tissue indicated that the accumulation of 239Pu in macrophages may be brought about in two ways. First, there may be phagocytosis of dead cells containing 239Pu. Second, 239Pu may follow the transfer of waste products of hormone synthesis from Leydig cells into macrophages. The significance of these observations is discussed with regard to the deposition of 239Pu in human testis.     

The cytotoxic activity of natural killer cells exposed to the joint and separate actions on the body of 239Pu, hexachlorobutadiene and tributyl phosphate

Wistar rats were subjected to a single exposure to 239Pu nitrate through inhalation with the subsequent procedure of imitation of inhalation or without it (the amount of 239Pu deposited in the lungs in 24 hr was 8.9 +/- 1.9 kBq/lung) and inhalation of hexachlorobutadiene and tributyl phosphate within one month in a combination with the radionuclide or without it. There was a 1.5-fold increase, above additive, in the harmful effect of 239Pu and chemical agents on the function of natural killers as observed 15--30 days after the combined exposure as compared to individual inhalation. On days 120 to 240 cell cytotoxic activity in rats of all groups was normalized to reach or to exceed the intact control.     

Cytogenetic mechanisms of aneuploidy in somatic cells of chemonuclear industry professionals with incorporated plutonium-239

Risks related to occupational exposure of humans to ionizing radiation remains a focus of attention. Of special importance is the poorly understood effect of ionizing radiation on the rate of numerical chromosome aberrations in human somatic cells. The purpose of this study was characterization of a radiation-induced increase in the rate of aneuploidy induced by incorporated plutonium-239 (²³⁹Pu) in somatic cells of chemonuclear industry workers. Significant differences in the chromosome 2, 7, 8, 12, and Y aneuploidy rate have been found between subjects with incorporated ²³⁹Pu and control subjects have been demonstrated. Chromosome nondisjunction has been found to be the main mechanism of aneuploidy induced by incorporated ²³⁹Pu.     

Structural genomic damages in workers of plutonium production

The research objective is assessment of structural genomic damages in plutonium workers. The study group included the Mayak nuclear workers subject to chronic occupational exposure to incorporated 239Pu and/or external gamma-rays. The analysis was performed based on the culture of lymphocytes in peripheral blood. The yield of intra-chromosomal exchange aberrations of chromosomal type on stained slides was analyzed using in situ fluorescent hybridization, mBAND. Linear relationships were revealed between (a) the total yield of chromosomal type aberrations (e.g. intra- and inter-chromosomal ones) and an absorbed dose from external exposure of the red bone marrow to gamma-rays, an absorbed dose from internal exposure to a-radiation from incorporated 239Pu; and (b) the yield of intra-chromosomal exchange aberrations of chromosomal type and an absorbed dose from exposure of the red bone marrow to 239Pu and 239Pu body burden.     

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.     

Repeated inhalation exposure of rats to aerosols of 144CeO2. I. Lung, liver, and skeletal dosimetry.

To develop a better understanding of the influence of cumulative radiation dose and dose rate to the lungs on the biological responses to inhaled radionuclides, several studies are in progress at this institute in which laboratory animals have been exposed once or repeatedly to aerosols of insoluble particles containing 144Ce or 239Pu. In the study reported here, F344 rats were exposed repeatedly to aerosols of 144CeO2 beginning at 94 days of age to reestablish desired lung burdens of 1.9, 9.2, 46, or 230 kBq of 144Ce every 60 days for 1 year (seven exposures). Other 94-day-old rats were exposed once to achieve similar desired initial lung burdens of 144Ce. Older rats were exposed once to achieve desired initial lung burdens of 46 or 230 kBq when 500 days of age, the age of the repeatedly exposed rats when exposed for the last time. Control rats were either unexposed, sham-exposed once or repeatedly, or exposed once or repeatedly to stable CeO2. Approximately equal numbers of male and female rats were used. The cumulative beta-radiation doses to the lungs, liver, and skeleton of rats exposed repeatedly were similar to those of rats with similar total lung burdens of 144Ce from a single inhalation exposure. The average beta-radiation dose rate to the lungs of the rats exposed repeatedly was about one-fifth of that in rats with similar total lung burdens after a single exposure.     

The antibody-producing cell count of rats exposed to polymeric 239Pu

In experiments with Wistar rats a study was made of the content of antibody-forming cells (AFC) in the spleen at remote times (3 to 12 months) after intravenous injection of 239Pu(IV) in doses of 166, 55, and 18 kBq/kg body mass. The doses absorbed in the central and peripheral immunity organs were defined. Pronounced spleen hypoplasia and profound inhibition of humoral immunity were displayed 1 year after the injection of a small amount of the radionuclide. AFC deficiency in animals was amounted to 11-32 per cent throughout the entire period of observation.     

Structural genomic damage in plutonium workers

The research objective is assessment of structural genomic damages in plutonium workers. The study group included workers of the Mayak Production Association subject to chronic occupational internal exposure to incorporated ²³⁹Pu and/or external γ-rays. A lymphocyte culture of peripheral blood was chosen as an object of study. The yield of intrachromosomal exchange aberrations of chromosomal type on stained slides was analyzed using fluorescent in situ hybridization, mBAND. Linear relationships were revealed between (a) the total yield of chromosome-type aberrations (intra- and inter-chromosomal ones) and the absorbed dose from external exposure of the red bone marrow (RBM) to γ rays, the absorbed dose from internal exposure of the RBM to α-radiation from incorporated ²³⁹Pu, and ²³⁹Pu body burden, and (b) the yield of intrachromosomal aberrations and an absorbed dose from internal exposure of the RBM to ²³⁹Pu and ²³⁹Pu body burden.     

Influence of dose rate on survival time for 239PuO2-induced radiation pneumonitis or pulmonary fibrosis in dogs.

Beagle dogs were exposed once or repeatedly to 0.75-microns-diameter monodisperse aerosols of 239PuO2 by pernasal inhalation. The dogs that were exposed once received alveolar depositions (+/- standard deviation) of 3.9 +/- 1.9 kBq/kg body mass and accumulated doses of 23 +/- 8 Gy to the lung before death at 5.4 +/- 1.7 years after exposure. Dogs exposed repeatedly received a total alveolar deposition of 5.3 +/- 0.9 kBq/kg body mass during 7 to 10 semiannual exposures and accumulated doses of 22 +/- 5 Gy to the lung before death at 4.9 +/- 0.7 years after first exposure. Clearance of the plutonium from the lung in the dogs exposed repeatedly was slower than in the dogs exposed once. All dogs in the repeated-exposure study and all but one dog in the single-exposure study died from radiation effects. Pulmonary fibrosis accounted for 72% of the radiation-related deaths in the single-exposure study and 87% in the repeated-exposure study. The remaining dogs died with pulmonary cancer. Based on total cumulative radiation dose, the times after exposure to death from radiation pneumonitis and pulmonary fibrosis were not significantly different for single and repeated exposures. Thus dose rate does not appear to be an important factor in predicting death from radiation pneumonitis or pulmonary fibrosis for dogs inhaling 239PuO2.     

Solubility of airborne radioactive fuel particles from the Chernobyl reactor and implication to dose

Airborne particles of nuclear fuel from the Chernobyl reactor that had been collected on air filters and stored, were characterised using in vitro dissolution tests to assess effective doses after their inhalation. As solvent, the Gamble biological fluid was used to simulate lung fluid. The solubility of the measured radionuclides decreased in the order ¹³⁷Cs>⁹⁰Sr>>²⁴¹Am^239+240Pu in the simulated lung fluid. The dissolution rate constant of e.g. ^239+249Pu ranged from 0.72 to 5.4×10^–6 g·cm^–2 d^–1 and decreased (for all nuclides) with increasing particle size as predicted from theoretical considerations. Considering the inhalation dose, decreasing dose with size and increasing doses with lower solubility may partly counterbalance each other for ¹³⁷Cs and ⁹⁰Sr. On the other hand, for ²³⁹Pu and ²⁴¹Am larger particles and associated lower solubility both change the resulting dose in the same direction towards lower values. The comparison of the experimentally determined dose coefficients with ICRP values indicates that nuclear fuel particles closely resemble type M material characteristics for ¹³⁷Cs and ⁹⁰Sr and type S material characteristics for ²³⁹Pu and ²⁴¹Am.     

Efficacy of polymeric 239Pu removal by liposome-encapsulated pentacin

Liposome-incapsulated pentacine (DTPA) removes intracellular polimeric 239Pu and colloidal hydroxide of polymeric 239Pu from a rat body in the amounts 2- and 4 times, respectively, exceeding those eliminated by free DTPA. However the amount of 239Pu removed decreases sharply with increasing 239Pu hydrolysation and polymerization. It is concluded that the effectiveness of 239Pu removal depends on the physico-chemical status of the radionuclide and its interaction with the biosubstrate rather than on the amount of DTPA entering the cells.     

Alkaline phosphatase and transaminase activity in the liver and blood serum of rats in the late periods following combined exposure to external gamma radiation and alpha radiation from plutonium-239

A study was made of activity of alkaline phosphatase and alanine- and aspartate aminotransferase in rat liver and blood serum at remote times after external gamma-irradiation combined with internal exposure to 239Pu nitrate delivered in two chronically effective doses. The radionuclide was shown to be mainly responsible for the changes observed in activity of the enzymes under study. The degree to which the changes were manifest depended upon dose of plutonium administered.     

Plutonium in the tissues of foetal, neonatal and suckling mice after Pu-administration to their dams.

Twelve-week-old female (C3H x 101)F1 mice were injected intravenously with an ultrafiltered solution of 239Pu in per cent trisodium citrate, and mated to uninjected PCT males. The plutonium content was examined radiochemically and autoradiographically in placentae and foetuses on the 12th and 18th days of gestation, and in neonates during the 24 hours after birth and also at 18 days postnatally. Plutonium was distributed in most tissues of the late foetus and the suckling as it is in adult mice. However, on both the 12th and 18th days of gestation the concentration in the yolk-sac splanchnopleure was much higher than in any other foetal tissue. The amount of 239Pu in 18-day-old sucklings was between two and seven times as great as in 1-day-old neonates because of ingestion of milk from the lactating dams. In the first litter following administration of the radionuclide to the dam, about 0.02 per cent of the plutonium injected was transferred to an individual offspring by the time of birth, and a further 0.08 per cent by the time of weaning.