Statistical modeling of carcinogenic risks in dogs that inhaled 238PuO2.

Combined analyses of data on 260 life-span beagle dogs that inhaled 238PuO2 at the Inhalation Toxicology Research Institute (ITRI) and at Pacific Northwest National Laboratory (PNNL) were conducted. The hazard functions (age-specific risks) for incidence of lung, bone and liver tumors were modeled as a function of cumulative radiation dose, and estimates of lifetime risks based on the combined data were developed. For lung tumors, linear-quadratic functions provided an adequate fit to the data from both laboratories, and linear functions provided an adequate fit when analyses were restricted to doses less than 20 Gy. The estimated risk coefficients for these functions were significantly larger when based on ITRI data compared to PNNL data, and dosimetry biases are a possible explanation for this difference. There was also evidence that the bone tumor response functions differed for the two laboratories, although these differences occurred primarily at high doses. These functions were clearly nonlinear (even when restricted to average skeletal doses less than 1 Gy), and evidence of radiation-induced bone tumors was found for doses less than 0.5 Gy in both laboratories. Liver tumor risks were similar for the two laboratories, and linear functions provided an adequate fit to these data. Lifetime risk estimates for lung and bone tumors derived from these data had wide confidence intervals, but were consistent with estimates currently used in radiation protection. The dog-based lifetime liver tumor risk estimate was an order of magnitude larger than that used in radiation protection, but the latter also carries large uncertainties. The application of common statistical methodology to data from two studies has allowed the identification of differences in these studies and has provided a basis for common risk estimates based on both data sets.

Biological effects of inhaled 238PuO2 in beagles.

Beagle dogs exposed to 238PuO2 aerosols (136 dogs, 13-22 per group, mean initial lung depositions of 0.0, 0.13, 0.68, 3.1, 13, 52 and 210 kBq) were observed throughout life to determine tissues at risk and dose-effect relationships. The pulmonary retention of 238Pu was represented by the sum of two exponentially decreasing components of the initial lung deposition; about 84% cleared with a 174-day half-time; the half-time of the remainder was 908 days. The average percentages of final body burden found in lung, skeleton, liver and thoracic lymph nodes in the 30 longest-surviving dogs (mean survival 14 years) were 1, 46, 42 and 6%, respectively. Of 116 beagles exposed to plutonium, 34 (29%) developed bone tumors, 31 (27%) developed lung tumors, and 8 (7%) developed liver tumors. Although lungs accumulated a higher average radiation dose than skeleton, more deaths were due to bone tumors than to lung tumors. Deterministic effects included radiation pneumonitis, osteodystrophy, hepatic nodular hyperplasia, lymphopenia, neutropenia and sclerosing tracheobronchial lymphadenitis. Hypoadrenocorticism was also observed in a few dogs. Increased serum alanine aminotransferase, indicative of liver damage, was observed in groups with > or =3.1 kBq initial lung deposition. Estimates of cumulative tissue dose in a human exposed to airborne 238PuO2 for 50 years at a rate of one annual limit on intake each year were derived based on a comparison of the data on metabolism for humans and beagles. The 50-year dose estimates for humans are an order of magnitude lower than doses at which increased incidence of neoplasia was observed in these dogs, whereas the projected doses to humans from 50-year exposure at the annual limit of intake are of similar magnitude to those at which deterministic effects were seen in the beagles.

Hypoadrenocorticism in beagles exposed to aerosols of plutonium-238 dioxide by inhalation.

Hypoadrenocorticism, known as Addison's disease in humans, was diagnosed in six beagles after inhalation of at least 1.7 kBq/g lung of 238PuO2. Histological examination of adrenal gland specimens obtained at necropsy revealed marked adrenal cortical atrophy in all cases. Autoradiographs showed only slight alpha-particle activity. Although the pathogenesis of adrenal cortical atrophy in these dogs is unclear, there is evidence to suggest an autoimmune disorder linked to damage resulting from alpha-particle irradiation to the lymphatic system.

Hepatic effects of inhaled plutonium dioxide in beagles.

The effects of inhaled 238PuO2 deposited in the liver of dogs were studied in beagles exposed to initial lung depositions ranging from 5.7 to 2979.7 Bq/g lung. Approximately 20% of the initial lung deposition was translocated to the liver by 1500 days after exposure. Life-span observations revealed that the liver contained 40% of the final body burden of plutonium, second only to the skeleton. Elevated serum liver enzyme activities were observed in dogs with final liver depositions of > or = 0.4 Bq/g, cumulative dose to the liver of > or = 0.18 Gy and annual dose rate > or = 0.02 Gy/year. Enzyme elevations were seen at one dose level lower than that in which bone or lung tumors were observed. Linear regression analysis revealed strong to moderate correlation between cumulative dose and dose rate and time to observed increases in liver enzyme activities. Liver tumors were late-occurring neoplasms observed at lower exposure levels where life span was not shortened by lung and bone tumors.

Examination of testicular tumours in the beagle dog exposed to inhaled plutonium.

Gross and light microscopic features of testicular neoplasms were examined in the male beagle dog used in three studies to examine the life-span effects of inhaled plutonium (Pu). One hundred and sixty-six cases of testicular neoplasia (TN) occurred among 105 dogs that ranged in age from 7.5 to 17.7 years at the time of diagnosis. The 166 cases of TN comprised 113 interstitial cell tumours, 27 seminomas in situ, 19 seminomas, and seven Sertoli cell tumours. Serum testosterone and estradiol 17-beta concentrations, and the serum testosterone-to-oestradiol ratio were determined in 39 dogs with TN and in five clinically normal, sexually intact, age-matched cohorts. Serum hormone concentrations did not differ significantly among tumour types or between dogs with neoplasms and age-matched cohorts. There was a significant relationship between initial lung deposition (ILD) of Pu and activity in the testis (Bq/g testis). The slope of the relationship was 0.35, 0.89 and 0.91 for 239PuO2, 238PuO2 and 239Pu(NO3)4 respectively. Pu in the testis at long times (> 5 years) after inhalation was between 0.0001 and 0.03% ILD, depending on the physicochemical form of Pu. Although the mean activity of Pu in the testis of dogs was higher in those life-span studies employing 238PuO2 and 239Pu(NO3)4, the cumulative proportion of dogs with tumours, the distribution of tumour types, and mean time to first tumour was not significantly different among the three studies or dose groups, including controls, within a study.

Hematological effects of inhaled plutonium dioxide in beagles.

A life-span study indicated that plutonium activity in the thoracic lymph nodes is a contributor to development of lymphopenia in beagles exposed to 239PuO2. Significant lymphopenia was found in 67 (58%) beagles given a single nose-only exposure to 239PuO2 to result in mean initial lung depositions ranging from 0.69 to 213.3 kBq. Lymphoid atrophy and sclerosis of the thoracic lymph nodes and lymphopenia were observed in exposure-level groups with initial lung depositions > or = 2.5 kBq. Those dogs with final plutonium concentrations in the thoracic lymph nodes > or = 0.4 kBq/g and dose rates > or = 0.01 Gy/day developed lymphopenia. Marked differences existed between chronically lymphopenic dogs and intermittently lymphopenic dogs with regard to initial lung deposition, time to lymphopenic events and absolute lymphocyte concentrations. Linear regression analysis revealed moderate correlation between reduction in lymphocyte values and initial lung deposition, in both magnitude and time of appearance after exposure. Cumulative dose and dose rate appeared to act together to produce initial effects on lymphocyte populations, while dose rate alone appeared to be responsible for the maintenance and subsequent cycles of lymphopenia seen over the life span. No primary tumors were associated with the thoracic lymph nodes in this study, although 70% of the lymphopenic dogs developed lung tumors.

Radiographically determined growth dynamics of primary lung tumors induced in dogs by inhalation of plutonium.

Beagles were exposed to aerosols of 239PuO2, 238PuO2, or 239Pu(NO3)4. Exponential growth constants for 50 primary lung tumors (23 bronchioloalveolar carcinomas, 22 papillary adenocarcinomas, 5 adenosquamous carcinomas) were calculated in 37 dogs, using sequential thoracic radiography. A wide range in doubling time (6 to 287 days) was observed. Mean +/- SEM doubling time was 93 +/- 10 days for bronchioloalveolar carcinoma, 107 +/- 13 days for papillary adenocarcinoma, and 101 +/- 36 days for adenosquamous carcinoma. Lung tumor growth rate in dogs was comparable to that in human patients with similar histologic tumor types. Linear regression analysis revealed significant (P < or = 0.0001) correlation between doubling time and survival of individual dogs. Doubling time was not significantly dependent on tumor type, sex, age at time of diagnosis, initial lung deposition, or isotope. Extrapolating time to tumor onset from tumor doubling time cannot be used to reliably predict the onset of malignancy.

Primary pulmonary chondrosarcoma in a dog.

Extraosseous chondrosarcomas are uncommon in the dog, and those originating in the lung are rare. This report presents a 9-year-old Beagle dog with a pulmonary mass which caused depression, fever, tachypnea, cough, and laboratory abnormalities. The mass was composed predominantly of chondroid tissue, and was histologically diagnosed as chondrosarcoma.

Time-related factors in the study of risks in animals and humans.

Data from epidemiological studies of humans exposed to potentially harmful substances are usually analyzed using methods that account for the dependence of risks on time-related factors such as age and follow-up period. Recently developed statistical procedures allow modeling of the age-specific risks as a function of dose as well as factors such as age at exposure, time since exposure, exposure duration, and dose rate. These procedures potentially allow more rigorous inferences and clearer understanding of the patterns of risk observed in epidemiological studies than has been available in the past. Statistical procedures that consider time-related factors can also be applied to laboratory animal data, providing information that is useful for the problems involved in extrapolating from animal studies to humans. By applying such procedures to data on exposure to the same substance in different species (including humans) or to different substances in the same species, better understanding of the relationship of risks across species and across substances can be achieved. In addition, such statistical procedures allow appropriate treatment of exposure that is accumulated over time and lead to improved understanding of patterns of risk over time. The approach is illustrated using data from a lifespan study of beagle dogs exposed to inhaled Pu.

A novel class of unstable 6-thioguanine-resistant cells from dog and human kidneys.

Thioguanine-resistant primary clones were grown from single cell suspensions obtained from dog and human kidneys by enzymatic digestion. In medium containing a relatively high concentration (10 micrograms/ml) of thioguanine, thioguanine-resistant primary clones arose from each source at frequencies ranging from 10(-4) to 10(-5). A reduction in total hypoxanthine uptake was found in the thioguanine-resistant primary clones which had developed in thioguanine medium, consistent with a reduction in hypoxanthine phosphoribosyltransferase activity. When these thioguanine-resistant primary clones were subsequently grown in the absence of thioguanine and assayed for the thioguanine-resistant phenotype and hypoxanthine phosphoribosyltransferase activity, it was found that most were now thioguanine-sensitive and yielded cell-free extracts with substantial amounts of hypoxanthine phosphoribosyltransferase activity. In contrast, thioguanine-resistant human clones grown continuously in the presence of thioguanine yielded cell-free extracts with little or no detectable hypoxanthine phosphoribosyltransferase activity. Southern blot analysis demonstrated no structural alterations in the hypoxanthine phosphoribosyltransferase gene in thioguanine-resistant primary human kidney clones. These results suggest that a novel mechanism(s) for thioguanine resistance and the control of hypoxanthine phosphoribosyltransferase expression may occur in dog and human kidney cells.

A method for measuring respiratory volume parameters of large animals during exposure to aerosols.

The respiratory volume monitor was specifically designed to aid in quantitative control of exposures of beagle dogs to aerosols without adversely affecting the quality of the aerosol. The system monitors and displays tidal volume, total inhaled volume, breath count and elapsed time of exposure. The error in the total inhaled volume measurement does not exceed 10%.

Clinical and pathological effects of cigarette smoke exposure in beagle dogs.

To compare the clinical and pathological effects of high and low nicotine cigarette smoke 12 young adult male beagles were separated into four equal groups and exposed to smoke from high (4.6 mg) or low (1.4 mg) nicotine cigarettes, administered in six or 12 cigarettes per day. Two control groups, sham-exposed and nontracheostomized, consisted of three dogs each. The dogs were exposed seven days per week for five months. Tracheobronchitis developed in smoke-exposed dogs; gross lesions were generally confined to the lungs and tracheobronchial lymph nodes. Histopathological changes were found in all smoke-exposed dogs, with slightly more severe or extensive lesions in the dogs exposed to 12 cigarettes per day. The incidence and severity of rhinitis, turbinate basal epithelial cell hyperplasia, and squamous metaplasia were increased among dogs in the high nicotine cigarette groups.

Focal mastocytosis in lymph nodes from a Beagle dog.

An 11-year-old Beagle dog had focal mastocytosis in the midtracheobronchial and left tracheobronchial lymph nodes. The cells were well-differentiated and were arranged in well-defined follicles. There were neither skin tumors nor mast-cell accumulations in other tissues.

Bone tumors induced by inhalation of 238PuO2 in dogs.

Plutonium-238, an alpha-emitting radionuclide, is used as a heat source in thermoeleltric power generators such as have been employed on lunar expeditions of communications satellites and in cardiac pacemakers. It has an 86.4 year half-life and emits 5.5 MeV alpha particles. Beagle dogs were given single 10-30 minute exposures to 238PuO2 aerosols to study the long-term translocation of plutonium and biological effects. Dogs with a terminal body burden ranging from 7-260 muCi were euthanized due to respiratory insufficiency related to plutonium-induced pneumonitis during the first 3 years after exposure. Nine of the 11 dogs euthanized during the 4-6 year postexposure period had osteosarcomas. The terminal plutonium body burden in the tumor-bearing dogs ranged from 0.5-2.6 muCi with 30-55% of the plutonium in the skeleton. Experiments are in progress to further define the dose-effect relationship of inhaled 238PuO2 and investigate the mechanisms of plutonium-induced neoplasia.