Nancy A. Gillett

Toxicity of inhaled plutonium dioxide in beagle dogs.

This study was conducted to determine the biological effects of inhaled 238PuO2 over the life spans of 144 beagle dogs. The dogs inhaled one of two sizes of monodisperse aerosols of 238PuO2 to achieve graded levels of initial lung burden (ILB). The aerosols also contained 169Yb to provide a gamma-ray-emitting label for the 238Pu inhaled by each dog. Excreta were collected periodically over each dogs life span to estimate plutonium excretion; at death, the tissues were analyzed radiochemically for plutonium activity. The tissue content and the amount of plutonium excreted were used to estimate the ILB. These data for each dog were used in a dosimetry model to estimate tissue doses. The lung, skeleton and liver received the highest alpha-particle doses, ranging from 0.16-68 Gy for the lung, 0.08-8.7 Gy for the skeleton and 0.18-19 for the liver. At death all dogs were necropsied, and all organs and lesions were sampled and examined by histopathology. Findings of non-neoplastic changes included neutropenia and lymphopenia that developed in a dose-related fashion soon after inhalation exposure. These effects persisted for up to 5 years in some animals, but no other health effects could be related to the blood changes observed. Radiation pneumonitis was observed among the dogs with the highest ILBs. Deaths from radiation pneumonitis occurred from 1.5 to 5.4 years after exposure. Tumors of the lung, skeleton and liver occurred beginning at about 3 years after exposure. Bone tumors found in 93 dogs were the most common cause of death. Lung tumors found in 46 dogs were the second most common cause of death. Liver tumors, which were found in 20 dogs but were the cause of death in only two dogs, occurred later than the tumors in bone and lung. Tumors in these three organs often occurred in the same animal and were competing causes of death. These findings in dogs suggest that similar dose-related biological effects could be expected in humans accidentally exposed to 238PuO2.

Effects of Protraction of the α 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.

Sequential analysis of the pathogenesis of plutonium-induced pulmonary neoplasms in the rat : morphology, morphometry, and cytokinetics

Light microscopy, morphometry, and cytokinetic techniques were used to examine the dynamics of plutonium-induced pulmonary proliferative lesions and neoplasms in rats at several intervals to 450 days after inhalation exposure to aerosols of 239PuO2. Maximal increases in alveolar and bronchiolar epithelial cell labeling were seen at 30 days; decreasing subsequently, the levels remained elevated above control indices. Focal proliferative epithelial lesions developed in the lung by 180 days and before the onset of pulmonary neoplasms. Pulmonary neoplasms, predominantly adenocarcinomas and squamous cell carcinomas, were initially observed at 308 days. The proliferative lesions progressed through a succession of morphological changes leading to the development of neoplasms. The volume density (fraction) and epithelial surface area of foci of alveolar epithelial hyperplasia increased progressively between 180 and 450 days after exposure, in contrast to the other proliferative lesions. We conclude that plutonium-induced pulmonary neoplasms develop through a succession of focal proliferative lesions that represent developmental preneoplastic lesions. Progressive increases in volume and epithelial surface area of the alveolar epithelial hyperplasias suggest that they may be more at risk for neoplastic transformation than the other histological types of proliferative foci.

Particle distribution in lung and lymph node tissues of rats and dogs and the migration of particle‐containing alveolar cells in vitro

Whole rat lungs and individual dog lung lobes were instilled either with low numbers (10(7)) or high numbers (10(9)) of fluorescent polystyrene microspheres (PLM), or with saline alone. Particle distributions in dog and rat lung lobes and tracheobronchial lymph nodes (TBLN) were studied up to several weeks after particle instillations using methacrylate-embedded tissues and epifluorescence light microscopy. Free alveolar cells were obtained from rats and dogs by lung lavage 1 or 7 d after particle instillations. Lavaged cells were tested for directed migration toward the chemoattractant N-formylmethionyl-leucyl-phenylalanine (FMLP). Random migration in the absence of the FMLP was used as a control. The dog lung interstitium contained many more particles than did the rat lung interstitium, and particle numbers in interstitial and TBLN cells of dogs were higher than in those of rats. FMLP enhanced the number of migrating cells about twofold. Increasing particle numbers in lavaged phagocytes (greater than 10 particles/phagocyte in dogs; greater than 20 particles/phagocyte in rats) decreased their ability to migrate. The higher fractions of particles in the dog lung interstitium are thought to be an important reason for prolonged retention and increased TBLN transport of deposited particles in dogs as compared with rats. Our results suggest that cell mobility is lost after ingestion of high numbers of particles, and that this occurs earlier with dog than with rat lung cells.

Dose responses from inhaled monodisperse aerosols of 244Cm2O3 in the lung, liver and skeleton of F344 rats and comparison with 239PuO2

The purpose of this study was to obtain information on the alpha-particle dose-response relationship of 244Cm in rats. Rats were exposed briefly by inhalation to graded levels of monodisperse aerosols of 244Cm2O3 heat-treated at 1150 degrees C. The initial lung burden (ILB) of each animal was determined by the use of the gamma-ray-emitting radionuclide 243Cm in the aerosols. Seven groups of 84-day-old F344/Crl rats (a total of 637 males and 645 females) were exposed once to 244Cm2O3 or sham-exposed to filtered ambient air. Mean ILBs of all rats per group ranged from 0.51 +/- 0.17 (+/-SD) to 240 +/- 82 kBq kg-1 body weight. Mean lifetime alpha-particle doses to the lungs per group ranged from 0.20 +/- 0.069 (+/-SD) to 36 +/- 6.5 Gy. After death, each rat was radiographed and necropsied. Dose-related increases occurred in incidences of benign and malignant lung neoplasms, except for the groups of rats with higher mean ILBs that were examined histologically (98 +/- 18 and 240 +/- 77 kBq kg-1 body weight) in which survival was markedly decreased. Also, average alpha-particle doses of 0.0014 +/- 0.00058 (+/-SD) to 0.17 +/- 0.091 Gy and 0.18 +/- 0.007 to 1.6 +/- 1.1 Gy were also absorbed by the liver and skeleton, respectively, in the rats in the different exposure groups. Primary liver neoplasms occurred in several rats. However, the incidence of these lesions was not related to dose. Increased incidences of bone neoplasms occurred only in rats receiving higher doses to the skeleton. Excess numbers of rats with lung neoplasms per 10(4) Gy to the lung per group ranged from 760 +/- 430 (+/- SE) at a mean dose of 0.48 Gy to 84 +/- 16 at a mean dose of 37 Gy. Risk factors for the lowest and highest ILB kg-1 body weight groups were not considered reliable because of large errors associated with these calculations and the life-span shortening in the highest ILB kg-1 group. Inhaled 244Cm2O3 appeared to be about 50% less effective as a lung carcinogen in rats compared to 239PuO2 at similar doses.

Expression of transforming growth factor alpha in plutonium-239-induced lung neoplasms in dogs: investigations of autocrine mechanisms of growth

We have previously shown that 47% of radiation-induced lung neoplasms in dogs exhibit increased expression of epidermal growth factor receptor (EGFR). In this study, we investigated the expression of transforming growth factor alpha (TGF-alpha), a ligand for EGFR, to determine if an autocrine mechanism for growth stimulation was present in these tumors. As determined by immunohistochemistry, 59% (26/44) of the lung neoplasms examined had increased expression of TGF-alpha. Expression of TGF-alpha was not related to the etiology of the tumor, e.g., spontaneous or plutonium-induced; however, it was related to the phenotype of the tumor. Statistical analysis of the correlation of EGFR and TGF-alpha expression within the same tumor did not show a positive association; however, specific phenotypes did have statistically significant expression of EGFR or TGF-alpha, suggesting that overexpression of either the ligand or its receptor conferred a growth advantage to the neoplasm. Twenty-seven percent (32/117) of radiation-induced proliferative epithelial foci expressed TGF-alpha, and a portion of those foci (8/32) expressed both EGFR and TGF-alpha. This supports the hypothesis that these foci represent preneoplastic lesions, and suggests that those foci exhibiting increased expression of the growth factor or its receptor are at greater risk for progressing to neoplasia.

Single inhalation exposure to 90SrCl2 in the beagle dog: hematological effects.

The toxicity of 90Sr administered by the inhalation route was studied in young adult Beagle dogs exposed once to aerosols containing 90SrCl2. Due to its relatively soluble chemical form, 90Sr was rapidly translocated from lung to bone where a substantial portion was retained for a long period of time. This resulted in only a brief radiation exposure of the respiratory tract and a protracted exposure of the skeleton. The long-term retained burdens ranged from 0.037 to 4.4 MBq 90Sr/kg body wt. Dogs were subsequently observed throughout their life span. Six dogs with long-term retained burdens of 1.7 to 4.1 MBq 90Sr/kg died at less than 32 days after exposure from radiation-induced bone marrow hypoplasia. Review of hematological parameters of all dogs showed a similar, consistent, and dose-related pancytopenia in those animals having a long-term retained burden of greater than 0.37 MBq 90Sr/kg. Thrombocytopenia and neutropenia persisted in all exposed dogs through 1000 days after exposure. For reference purposes, a burden of 0.37 MBq 90Sr/kg is calculated to deliver an average radiation dose to the skeleton over 30, 100, and 1000 days after intake of 1.0, 2.8, and 17 Gy, respectively. The hematologic changes were similar to those seen in people exposed to high doses of whole-body external radiation.

Expression of transforming growth factor alpha and epidermal growth factor receptor in rat lung neoplasms induced by plutonium-239

Ninety-two rat lung proliferative lesions and neoplasms induced by inhaled 239PuO2 were evaluated for aberrant expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR). Expression of TGF-alpha protein, measured by immunohistochemistry, was higher in 94% of the squamous cell carcinomas and 87% of the foci of alveolar epithelial squamous metaplasia than that exhibited by the normal-appearing, adjacent lung parenchyma. In contrast, only 20% of adenocarcinomas and foci of epithelial hyperplasia expressed elevated levels of TGF-alpha. Many neoplasms expressing TGF-alpha also expressed excessive levels of EGFR mRNA. Southern and DNA slot blot analyses showed that the elevated EGFR expression was not due to amplification of the EGFR gene. These data suggest that increased amounts of TGF-alpha were early alterations in the progression of plutonium-induced squamous cell carcinoma, and these increases may occur in parallel with overexpression of the receptor for this growth factor. Together, these alterations create a potential autocrine loop for sustaining clonal expansion of cells initiated by high-LET radiation.