Effect of inhaled alpha-emitting nuclides on mouse alveolar macrophages

The effects of inhaled alpha emitters on the free cell population of the mouse lung were investigated up to 100 days after exposure. Groups of mice inhaled aerosols of 238PuO2, 239PuO2, or 241Am(NO3)3 to give alveolar deposits resulting in lung-averaged cumulative absorbed doses of about 20 Gy by the end of the study. Initially, with 238Pu most of the activity was associated with relatively few pulmonary alveolar macrophages (PAM), whereas with 241Am, all pulmonary alveolar macrophages were labeled and a substantial fraction was extracellular. The free cell population of the lung was sampled using bronchoalveolar lavage. The main parameters investigated were (a) the recovery and total numbers of free cells, including PAM, lymphocytes, and neutrophils; (b) the incidence of nuclear abnormalities in PAM (cells with more than one nucleus or with micronuclei); and (c) metabolic activation of PAM from measurements of their size and associated beta-glucuronidase activity. All three actinides produced depletions in total numbers of PAM, increased incidences of nuclear abnormalities, and metabolic activation of PAM, without a marked infiltration of inflammatory cells. Americium-241, which is distributed relatively uniformly in PAM, produced the most marked changes in that population and 238Pu, which gave the most inhomogeneous distribution of activity, produced the least.     

The development and interlobar distribution of plutonium-induced pulmonary fibrosis in mice

Six-week-old mice were exposed by inhalation to an aerosol of 239PuO2 (activity median aerodynamic diameter 2.2 microns) to establish mean alveolar depositions at 2 days after exposure of 4, 40, and 930 Bq of 239Pu. Animals were killed serially after 3, 6, 12, and 18 months at which times the development of the pulmonary fibrotic lesion was assessed by both biochemical and histopathological techniques. Individual measurements of both fresh and dry weights, protein, DNA, and hydroxyproline were made on whole lung and also on each of the five constituent lobes. Early and sustained increases in lung mass, lung protein, and total lung collagen were found, together with a depression of the total cellularity of the lung at 6 and 12 months after exposure. Although at later times compensatory hypertrophy of less affected areas distorted the relationship, systematic trends in the severity of responses between lobes were found. These trends were related to the initial lobar concentrations of 239Pu.     

Effects of the single or repeated inhalation exposure of Syrian hamsters to aerosols of 239PuO2

Male Syrian hamsters were scheduled to be exposed by inhalation approximately every 60 days for 1 year (7 exposures) to aerosols of 239PuO2 beginning at 84 days of age. Other hamsters were exposed once when 84 or 320 days of age. Plutonium-239 deposited in the lungs by the repeated inhalation exposures was cleared from the lungs at a rate similar to that following a single inhalation exposure. The incidence of radiation pneumonitis, bronchiolar epithelial hyperplasia, and alveolar squamous metaplasia were the only lesions that were related to radiation dose. Only two primary lung tumours were found among the hamsters exposed to 239PuO2. No primary lung tumours were found in the control hamsters. It was concluded that the incidence of lung tumours was not increased by the protraction of the alpha radiation dose to the lungs from repeated inhalation 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.     

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.     

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.     

Microdosimetry of rat alveolar type II cells irradiated with alpha particles from 239PuO2

The alveolar type II cell is one of the critical cells for radiation damage in the lungs after inhalation of radioactive aerosols. With the aid of a Quantimet-970 image analyzer and a VAX-11/780 computer, we calculated the radiation dose to rat alveolar type II cells from alpha particles emitted by 239PuO2. A series of dosimetric parameters for type II cells, including track length distribution, linear energy transfer (LET), values of the specific energy for a single hit of a spherical target (z1), cellular dose, hit number, and their spatial distributions were calculated. By comparing the volume density of type II cells and lung tissue with energy deposited in alveolar type II cells, we found that the energy deposited per unit volume of type II cells was larger than that of lung tissue excluding type II cells. The z1 for spherical targets and the LET across type II cells were less than those in lung tissue excluding type II cells. The age of the rat and damage to lung by inhalation may significantly influence some of the parameters. The neoplastic transformation probability for type II cells is also discussed. The results suggest that the type II cell is an important target cell in the rat lung for exposure to inhaled 239PuO2.     

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.     

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.     

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.     

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.     

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.     

Macrophage depletion of mouse lung following inhalation of 239PuO2

Changes in the free-cell population of the lungs of two strains of mice (SAS/4 and CBA/H) were studied up to 4 months after inhalation exposure to a sized fraction of 239PuO2 particles (1.5 micron AMAD) to give initial alveolar depositions (IADs) ranging from 17 to 810 Bq. A sample of the free-cell population of the lung was recovered by bronchoalveolar lavage, and a radiometric method was used to estimate the total number of pulmonary alveolar macrophages (PAM) in the lung. The response of the lung to 239PuO2 was characterized by an initial, dose-dependent depression in the total number of PAM following an IAD as low as 50 Bq. At IADs greater than 150 Bq, the initial depression continued for longer, merging into a chronic phase in which the PAM were larger and were accompanied by a minor infiltration of leukocytes. These findings were confirmed by histology, which also revealed focal accumulations of Type II pneumocytes. The results indicate that inhaled alpha-emitting particles are effective at producing a depletion in the alveolar macrophage population at relatively low IADs and that chronic effects on the cells can be produced by higher concentrations.     

Regulatory effects of eotaxin on acute lung inflammatory injury

Eotaxin, which is a major mediator for eosinophil recruitment into lung, has regulatory effects on neutrophil-dependent acute inflammatory injury triggered by intrapulmonary deposition of IgG immune complexes in rats. In this model, eotaxin mRNA and protein were up-regulated during the inflammatory response, resulting in eotaxin protein expression in alveolar macrophages and in alveolar epithelial cells. Ab-induced blockade of eotaxin in vivo caused enhanced NF-kappaB activation in lung, substantial increases in bronchoalveolar lavage levels of macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC), and increased MIP-2 and CINC mRNA expression in alveolar macrophages. In contrast, TNF-alpha levels were unaffected, and IL-10 levels fell. Under these experimental conditions, lung neutrophil accumulation was significantly increased, and vascular injury, as reflected by extravascular leak of (125)I-albumin, was enhanced. Conversely, when recombinant eotaxin was administered in the same inflammatory model of lung injury, bronchoalveolar lavage levels of MIP-2 were reduced, as was neutrophil accumulation and the intensity of lung injury. In vitro stimulation of rat alveolar macrophages with IgG immune complexes greatly increased expression of mRNA and protein for MIP-2, CINC, MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta. In the copresence of eotaxin, the increased levels of MIP-2 and CINC mRNAs were markedly diminished, whereas MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta expression of mRNA and protein was not affected. These data suggest that endogenous eotaxin, which is expressed during the acute lung inflammatory response, plays a regulatory role in neutrophil recruitment into lung and the ensuing inflammatory damage.     

Lung-specific delivery of cytokines induces sustained pulmonary and systemic immunomodulation in rats

The recombinant cytokines IFN-gamma and TNF-alpha stimulate several macrophage-mediated functions important in host defense. However, systemic administration of cytokines may be limited by significant host toxicity. We investigated whether aerosolized cytokines can stimulate alveolar macrophage and blood monocyte function, and whether they induce an inflammatory response in the lungs of normal rats. We found that aerosolized murine rIFN-gamma or recombinant human TNF-alpha increased IL-1 production by both alveolar macrophages and blood monocytes for at least 5 days after administration. Furthermore, murine rIFN-gamma increased the expression of Ia Ag on alveolar macrophages and human rTNF-alpha increased alveolar macrophage- and blood monocyte-mediated tumor lysis. Sequential aerosolization of IFN-gamma and TNF-alpha significantly increased both IL-1 release and Ia expression compared to either cytokine administered alone. Aerosolized human rTNF-alpha achieved lung levels comparable to those produced by an i.v. TNF-alpha dose reported to cause diffuse organ injury and death in rats. However, plasma TNF-alpha levels were several thousand-fold lower after aerosol administration. Aerosolized cytokines did not induce lung edema or an inflammatory cell infiltrate within the airways or alveoli. Aerosolized human rTNF-alpha alone, or murine rIFN-gamma and human rTNF-alpha, induced margination of leukocytes in pulmonary blood vessels 1 day after aerosolization, and a few small foci of pulmonary hemorrhage 5 days later. We conclude that aerosol administration of IFN-gamma or TNF-alpha enhances both pulmonary and systemic monocyte function, and that the combination of IFN-gamma and TNF-alpha produce additive or synergistic effects. Aerosolized cytokines induce only a minimal pulmonary inflammatory response. Aerosolized TNF-alpha produces high cytokine levels in the lung but very low uptake into the circulation.     

Promotion of pulmonary carcinogenesis by plutonium particle aggregation following inhalation of 239PuO2

Promotion of lung tumor formation from inhaled 239PuO2 in rats may be associated with aggregation of plutonium particles near bronchioles. The relationship of plutonium particle aggregation in the lung and the development of lung tumors after inhalation of 239PuO2 was studied in 664 life span rats with mean lung doses ranging from 0.35 to 20 Gy. Plutonium particle concentration and aggregation were determined from autoradiographic sections of the left lung lobe. The increase in particles/cm2 and mean number of particles per aggregate up to 20 Gy were directly proportional to lung dose. Aggregates with greater than 25 particles increased linearly with dose from 0.2% at 1.4 Gy to 8.2% at 20 Gy, in a pattern similar to increasing severity of pulmonary fibrosis and incidence of lung tumors. Lung tumor incidence increased from about 6% at 1.4 Gy to 83% at 8 Gy; no further increase in lung tumors was seen at doses greater than 8 Gy. Maximum lung tumor incidence at 8 Gy corresponded to a particle concentration of 130/cm2 and four particles/aggregate with 4% of aggregates having greater than 25 particles. Aggregation of inhaled plutonium particles in clusters of greater than 25 particles resulted in daily doses of only a few centigray to focal tissue regions containing clustered particles, yet these doses appeared sufficient to cause pulmonary fibrosis and promotion of pulmonary carcinogenesis.     

Effects of protraction of the alpha dose to the lungs of mice by repeated inhalation exposure to aerosols of 239PuO2

To determine the long-term biological effects of protracted alpha irradiation of the lung, 84-day-old C57BL/6J mice were repeatedly exposed by inhalation to aerosols of 239PuO2 every other month for up to six exposures in 10 months to reestablish lung burdens of 20, 90, or 460 Bq. Other mice were exposed only once when either 84 or 460 days of age to achieve desired initial lung burdens of 20, 90, 460, or 2300 Bq. Suitable control groups were maintained. Groups of mice with similar cumulative alpha doses to the lung had 3.4 to 4.4 times greater incidence of pulmonary tumors (adenomas and adenocarcinomas) when the dose to the lung was protracted by the repeated inhalation exposures compared to mice that received a single inhalation exposure. Excess pulmonary tumors per unit dose to the lung were also greater in groups of repeatedly exposed mice compared to those exposed only once. Repeatedly exposed mice also died earlier with pulmonary tumors than did those exposed once. It appears that protraction of an alpha dose to lungs increases the carcinogenic risk of inhaled 239PuO2 in mice.     

Cytokines and oxygen radicals after hyperoxia in preterm and term alveolar macrophages

To determine if the alveolar macrophage inflammatory cytokine response to oxygen differs in premature cells, macrophages were obtained from litters of premature (27 days) and term (31 days) rabbits. The majority of these cells were nonspecific esterase positive and actively phagocytosed latex particles. The cells that expressed cytokines also reacted with a monoclonal antibody against rabbit macrophages. After incubation overnight in 5 or 95% oxygen, the amount of interleukin (IL)-1beta and IL-8 mRNA was assessed by RT-PCR and the amount of cytokine protein by quantitative immunofluorescence microscopy. The preterm macrophage showed a significant increase in cytokine mRNA and protein after overnight incubation in 95% oxygen. This response was not seen in the term cells. Only premature macrophages had a significant increase in intracellular oxygen radical content, measured by 2',7'-dichlorofluorescin analysis, after incubation in 95% oxygen. This enhanced inflammatory cytokine response to oxygen may be one mechanism involved in the early development of chronic lung disease in premature infants.