Richard E. Weller

Biological Effects of Inhaled 239PuO2 in Beagles

Seven groups of 8–24 Beagle dogs, exposed to 239PuO2 aerosols by inhalation [mean initial lung depositions (ILD) of 0.0, 0.14, 0.63, 3.2, 13, 44 and 210 kBq] were observed throughout their lives to determine tissues at risk and dose-effect relationships. The mean average pulmonary retention half-time of 239Pu was 1,192 days. Most (70%) of the plutonium recovered at death in dogs surviving >10 years after exposure was found in the thoracic lymph nodes with ∼15% in lung, ∼10% in liver and ∼2% in bone. Eight dogs at the highest exposure levels died from radiation pneumonitis prior to a minimal 3-year latency period after exposure for the observation of lung tumors, with the first succumbing 337 days after exposure. Of 108 plutonium-exposed Beagles with ILD <100 kBq, 51 (47%) had lung tumors with significantly increased incidence in those dogs with total lung dose of ≥1.1 Gy at death. The primary non-neoplastic effects observed were lymphopenia, atrophy and fibrosis of the thoracic lymph nodes, radiation pneumonitis and pulmonary fibrosis, and bacterial pneumonia. Lesions of the thoracic lymph nodes were observed in 98 of 108 exposed dogs, but there were no primary neoplasms of the lymph nodes. Bacterial pneumonia was observed in 13 plutonium-exposed dogs and was the most notable non-neoplastic cause of death, with survival nearly the same as that of controls. Setting of dose limits on the basis of detrimental effects commonly considers and differentiates between stochastic and deterministic effects, raising the question of whether the non-neoplastic effects found in this study were deterministic. The International Commission on Radiation Protection (ICRP), National Council on Radiation Protection & Measurements (NCRP), and similar organizations generally consider effects that increase in incidence and severity to meet the definition of deterministic. We demonstrated the radiation dose-related nature of effects such as pneumonitis and fibrosis graphically and lymphopenia numerically, rather than by quantified estimates. It is clear, however, that both incidence and severity increased with ILD and radiation dose and should be considered as deterministic effects.

Neoplasia/Proliferative Disorders

Publisher Summary This chapter discusses neoplastic and proliferative disorders in nonhuman primates. The chapter also provides an overview of neoplasia in nonhuman primates for veterinary practitioners, laboratory animal veterinarians, and scientists concerned with the clinical management of the diseases and the comparative aspects of oncology. Neoplasms are classically divided into two primary groups: benign and malignant. The classification of neoplasia is based on many different criteria such as histogenesis, histological features, and biological behavior. The etiology of neoplasms in nonhuman primates is largely unknown, although several contributory causes have been identified in different species, including genetic factors, ionizing radiation, chemical carcinogens, and viruses. The development of neoplasia is a multistep process involving the clonal evolution of abnormal cell populations that gain a selective growth advantage over normal cells by accumulating specific alterations in at least two groups of genes: protooncogenes and tumor-suppressor genes. A description of various tumors—tumors of the skin, soft tissues, and bones; tumors of the cardiovascular system; tumors of lymphoid and hematopoietic systems; and tumors of the respiratory organs—is also provided in the chapter.

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.

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 (10μg/ 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 hypoxanth phosphoribosyltransferase expression may occur in dog and human kidney cells.

CARCINOGENESIS FROM INHALED 239PuO2 IN BEAGLES: EVIDENCE FOR RADIATION HOMEOSTASIS AT LOW DOSES?

From the early 1970s to the late 1980s, Pacific Northwest National Laboratory conducted life-span studies in beagle dogs on the biological effects of inhaled plutonium (238PuO2, 239PuO2, and 239Pu[NO3]4) to help predict risks associated with accidental intakes in workers. Years later, the purpose of the present follow-up study was to reassess the dose-response relationship for lung cancer in the 239PuO2 dogs compared to controls—with particular focus on the dose-response at relatively low lung doses. A 239PuO2 aerosol (2.3 &mgr;m activity-median aerodynamic diameter, 1.9 &mgr;m geometric standard deviation) was administered to six groups of 20 young (18-mo-old) beagle dogs (10 males and 10 females) by inhalation at six different activity levels, as previously described in Laboratory reports. Control dogs were sham-exposed. In dose level 1, initial pulmonary lung depositions were 130 ± 48 Bq (3.5 ± 1.3 nCi), corresponding to 1 Bq g−1 lung tissue (0.029 ± 0.001 nCi g−1). Groups 2 through 6 received initial lung depositions (mean values) of 760, 2,724, 10,345, 37,900, and 200,000 Bq (22, 79, 300, 1,100, and 5,800 nCi) 239PuO2, respectively. For each dog, the absorbed dose to lungs was calculated from the initial lung burden and the final lung burden at time of death and lung mass, assuming a single, long-term retention function. Insoluble plutonium oxide exhibited long retention times in the lungs. Increased dose-dependent mortality due to lung cancer (bronchiolar-alveolar carcinoma, adenocarcinoma, and epidermoid carcinoma) and radiation pneumonitis (in the highest exposure group) were observed in dogs exposed to 239PuO2. Calculated lung doses ranged from a few cGy (lowest exposure level) to 7,764 cGy in dogs that experienced early deaths from radiation pneumonitis. Data were regrouped by lifetime lung dose and plotted as a function of lung tumor incidence. The lung tumor incidence in controls and zero-dose exposed dogs was 18% (5/28). However, no lung tumors were observed in 16 dogs with the lowest lung doses (8 to 22 cGy, mean 14.4 ± 7.6 cGy), and only one lung tumor was observed in the next 10 dogs with lung doses ranging from 27 to 48 cGy (mean 37.5 ± 10.9 cGy). By least-squares analysis, a pure-quadratic function represented the overall dose-response (n = 137, r = 0.96) with no apparent dose-related threshold. Reducing this function to three linear dose-response components, we calculated risk coefficients for each. However, the incidence of lung tumors at zero dose was significantly greater than the incidence at low dose (at the p ≤ 0.053 confidence level), suggesting a protective effect (radiation homeostasis) of alpha-particle radiation from 239PuO2. If a threshold for lung cancer incidence exists, it will be observed in the range 15 to 40 cGy.

Spontaneous terminal ileitis resembling Crohn disease in captive tamarins

Five tamarins (four Saguinus mystax and one S. labiatus) died with wasting syndrome characterized by chronic diarrhea at the Center for Reproduction and Conservation of Non‐Human Primates in Iquitos, Peru. At necropsy, the terminal ileum of all affected tamarins was found to be markedly thickened. Histologically, the terminal ileal mucosa was completely ulcerated, and effaced by debris and mononuclear inflammatory cells. The submucosa and serosa were thickened by fibroplasia, mononuclear cell infiltrates and variable edema. No infectious agent was observed. The lesions were similar to those described previously for Crohn disease. This is to our knowledge the first report of terminal ileitis resembling Crohn disease in non‐human primates.

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

Hypoadrenocorticism, known as Addisons 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.

K-ras mutations in 239PuO2 canine lung neoplasms

Single-strand conformational polymorphism (SSCP) analysis and direct sequencing methods were used to examine lung tumors derived from a cohort of beagle dogs with inhalational exposures to 239PuO2. These exposures were done at Pacific Northwest Laboratories where 18-month-old beagle dogs were given 239PuO2 by single-dose inhalation and allowed to live out their life-spans. Formalin-fixed paraffin-embedded blocks of tissues from 25 dogs exposed to 239PuO2 by aerosol inhalation which later developed lung tumors were available for this study. Two of 25 tumors had mutations within exon 1 of K-ras detected by SSCP analysis. Both mutations were GGT to GAT transitions at codon 12 confirmed by direct sequencing experiments. One was an adenocarcinoma from the medium-high exposure group and the other was a broncheolo-alveolar carcinoma from the medium-low exposure group. The rate of K-ras mutations in plutonium-induced lung tumors described herein (8%) was greater than previously described in canine plutonium-induced lung tumors (0%), but was less than that which we have described in spontaneous canine lung cancer (16%), less than that reported for human spontaneous non-small cell lung cancer (13-36%) and less than that described in rats with spontaneous lung cancer (40%) or lung tumors following 239Pu inhalation exposure (46%).

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.