Contents
Research In Radiobiology – Annual Report 1986
Written by Charles W. Mays, Glenn N. Taylor and Walter Angus.
ABSTRACT
The general protocol is given for beagle studies at the Radiobiology Division of the University of Utah. Then each radionuclide toxicity study is summarized. Last, the numbers of injected and living beagles in the chronic toxicity studies are tabulated.
GENERAL PROTOCOL FOR RADIONUCLIDE INVESTIGATIONS
The Beagle studies at Utah were started by the Atomic Energy Commission in 1950 to predict the risk from 239Pu in people, based on the observed effects in the U.S. radium dial painters and the relative toxicity of 239Pu vs. 226Ra, to be established in young adult beagles. For simplicity and reproducibility, most of the dogs received a single intravenous injection of radionuclide, usually in citrate solution, at about 17 months of age when their skeletal maturity corresponded to that of an 18-year old radium dial painter or plutonium worker (Dougherty et al, 1962; Stover and Stover, 1972; also see the Introduction to the Appendix of this Progress Report, page A-1. Additional radionuclides were also studied in young adult beagles. Some dogs were injected with 239Pu or 226Ra at either 3 months of age (to represent children) or 5 years of age (to represent middle aged persons). Most dogs were confined in metabolism cages from 1 week before injection to 3 weeks after injection (for excreta collection). The exception was that the dogs receiving short-lived 224Ra in either 1, 10, or 50 injections were not confined, because their duration of injections extended up to almost 1 year.
Experimental dogs were designated as either toxicity or test animals. Toxicity dogs were allowed to live out their lifespans or until sacrifice was indicated for humane reasons, such as to prevent pain. To promote a long and healthy life, most tumors were removed surgically under anesthesia, except for bone cancers, which were not removed. Test dogs could be sacrificed as needed for special studies, such as dosimetry.
The dogs were housed, typically three to a kennel run that was 2.5 feet wide and 14 feet long (outside) and 4 feet long (inside). A swinging door permitted the dogs to go either inside or outside. The concrete floor of the kennel was hosed free of excreta 2-3 times daily, and in winter the kennel floor was heated to prevent snow accumulation. The dogs had continual access to tap water for drinking and were fed once per day at noon with about 0.4 pounds of dry kibble, moistened with water and supplemented with a little meat (Rehfeld et al, 1972). The dogs are immunized yearly against distemper, hepatitis, rabies, leptospirosis, and since about 1979 against parvovirus.
Each dog is examined daily by an animal handler and, if sick, treated by a veterinarian. If appropriate, the dog is isolated in the sick bay. Each dog receives a complete physical examination by a veterinarian every 6 months. This examination includes palpation of the lymph nodes, bone structures, abdominal cavity; observation of hair coat, general attitude, muscle tone, eyes, ears, oral cavity, teeth, perineal regions, auscultation of the thoracic cavity and heart; recording the pulse and respiratory rate.
All of the bone-seeking alpha-emitters studied have caused severe damage to the skeleton. Particular emphasis has been given to bone fractures, the location of osteosarcomas and the evaluation of tumor growth rates based on sequential radiographs of the expanding tumors (Thurman et al, 1971).
Because of concern that the dose from diagnostic x-rays might be com- parable to that from the lowest levels of injected radionuclides, the x-ray frequency for dogs at the 1-level and below was decreased to 1 series per year starting in 1957. Furthermore, the single series was abbreviated to two radiographs (left mandible and a right lateral of the right foreleg). This abbreviated procedure was later extended to the 1.7-level of 241Am only, due to the low deposition of 241 Am in bone. The higher level dogs continued to receive the full series of 10 radiographs twice a year. Radiographs are also taken when a bone tumor or fracture is discovered or suspected. Sequential radiographs of the bone tumors are taken for growth rate evaluation until sacrifice is clinically appropriate.
At autopsy, the bones are defleshed and postmortem x-rays showing two views of each bone are taken. These radiographs are used to identify possible tumor sites that are then examined histologically.
The radiation dose averaged over the skeleton is evaluated for each dog. When possible, this information is based on each animal’s measured retention. One line summaries are given in the Appendix of this Progress Report for each dog, showing the dog’s designation, age at injection, injection weight, injected microCi/kg, date injected, days from injection to death, average skeletal dose in rad, and comments on the dog’s diseases with special emphasis on cancer. The success of this format is evidenced by its subsequent use in the beagle studies at Albuquerque, Davis and Richland, as well as for the U.S. radium persons and for German patients injected with 224Ra.
For ease of understanding, each of the toxicity studies in young adult beagles will now be presented in the time sequence the radionuclides were first injected. Then the studies of 239pu and 226Ra injected into beagles at 3 months of age and at 5 years of age will be described.
TOXICITY STUDIES IN YOUNG ADULT BEAGLES
239pu, injected from 1952-1974
Initially the injected dosages ranged from 0.016 microCi/kg (the 1-level) where no harm was predicted, up by a sequence of levels to 2.86 microCi/kg (the 5-level) where severe injury occurred from hematological damage, bone fractures, and bone sarcomas (Dougherty et al. 1962). However, when an osteosarcoma occurred at the supposedly safe 1-level, several lower levels were introduced in 1964 down to 0.0006 microCi/kg (the 0.1-level) that resulted in an average skeletal dose of only 2 rads at death. Among the 28 dogs at this level, one developed a bone sarcoma and another an epidermoid carcinoma of the frontal sinus; both cancers probably induced by 239pu. The selective deposition of 239Pu on bone surfaces (Jee, 1972) makes 239Pu the most effective of any radionuclide studied at Radiobiology Uivision in bone sarcoma induction at low doses, per rad of average skeletal dose (Mays et al., 1987). Pu-239 also deposits throughout the liver (Stover et al., 1971) and induces liver cancers (Taylor et al., 1972).
226Ra, 1953-1970
226Ra enabled the relative toxicity of 239Pu vs. 226Ra to be estab- lished in beagles so that the known toxicity in the U.S. radium dial painters could be modified to predict the risk to humans from 239Pu-induced bone sarcoma. 226Ra is chemically similar to calcium and deposits through- out bone volume, especially in regions of active growth. The average skeletal dose for each dog is based on the measured retention of 226Ra and progeny (Lloyd et al., 1986). In beagles 226Ra produced bone fractures at high dosage. bone sarcomas were induced over a wide range of doses. These effects were also seen in the radium dial painters. But in beagles 226Ra is also avidly taken up by the melanocytes of the eye, inducing malignant melanoma (Taylor et al., 1972a), a type of cancer not seen in the radium dial painters, presumably due to a lower retention of radium by the human eye.
228Ra, 1954-1962
228Ra (MsTh) was included because it was received by many of the radium dial painters. In terms of average skeletal dose, 228Ra was about twice as effective in bone sarcoma induction as 226Ra (Lloyd et al., 1986a; Mays et al., 1987). The difference may be largely due to some 228Ra progeny partially redepositing on bone surfaces. 228Ra was even more effective than 226Ra in producing eye melanomas in beagles (Taylor et al., 1972a). An important spinoff from the study of 228Ra in dogs was the discovery that the physical half-period of 228Ra is 5.77 = or – 0.02 yr, not Lise Meitner’s earlier value of 6.7 yr (Mays et al., 1962). The correct half-period increased the calculated dose from 228Ra in the dial painters by about a factor of two.
228Th, 1954-1963
228Ra decays to 228Th and there was early concern that the intestinal absorption of the 228Th in dial paint might be high. Later, Maletskos et al. (1969) showed that absorption of 228Th from the human G.I. tract was low, about 0.02% compared to 20% for radium. However, the 228Th toxicity data from beagles proved very useful in evaluating the risk from radionuclides in the proposed Thorium Breeder Reactor (Lloyd et al., 1984).
90Sr, 1955-1966
90Sr toxicity was evaluated because of worldwide concern about radioactive fallout. Few effects were observed at average skeletal doses below 5000 rads, but bone sarcomas occurred frequently at higher doses. Most interesting was the relative ineffectiveness of 90Sr in producing leukemia in adult beagles (Dougherty et al., 1972). This agrees with the low frequency of myeloproliferative syndrome (MPS) in beagles at Davis, California, who ingested 90Sr as adults. However, a high incidence of MPS was observed in Davis beagles exposed to high dosage of 90Sr from fetal age to adulthood (Book et al., 1982).
241Am, 1966-1975
241Am was the first transplutonium radionuclide to be evaluated for toxicity in beagles at the U. of Utah (Mays and Dougherty, 1972). Because of strong interest in 241Am, especially by Charles Dunham, Head of the AEC’S Division of Biology and Medicine, the original test study was expand- ed into a full scale toxicity study with about 12 dogs per dosage level. Control dogs concurrently assigned to the low-level studies of 239Pu and 226Ra were considered suitable as controls for 241Am. In 1975, the number of beagles at the 1-level and 1.7-level were increased to 26 and 24 dogs, respectively, to investigate more extensively the induction of liver cancer by alpha-emitters. The liver retention of 241Am is higher than that for any other monomeric radionuclide studied in beagles at the University of Utah (Lloyd et al., 1984a).
249Cf, 1971-1974
249Cf, which emits alpha-particles in 100% of its decays, was the next transplutonium radionuclide to be investigated (Lloyd et al, 1975). Fortuitously, tracer amounts of beta-emitting 249Bk were present with the alpha-emitting 249Cf, making it possible to establish simultaneously that the microscopic depositions of berkelium (element 97) and californium (element 98) were similar (Taylor et al., 1972).
252Cf 1971-1973
252Cf releases half of its decay energy in alpha-particles and half in extremely densely ionizing fission fragments. The 252Cf and 249Cf studies were conducted simultaneously in beagles, and also in mice. In the mouse studies fission fragments were much less effective than alpha-particles, per rad of average skeletal dose, in bone sarcoma induction (Taylor et al., 1983). Already it is obvious that the fission fragment dose is much less effective than alpha-particle dose for bone sarcoma induction in the almost completed beagle studies with 252Cf and 249Cf. This information is of significance to the astronaut who may receive appreciable radiation dose from extremely densely ionizing cosmic rays.
253Es 1973-1974
Einsteinium (element 99) was the highest atomic number element to be investigated for radionuclide toxicity in beagles. Einsteinium appeared to resemble Cf most closely in its excretion, retention and tissue distribution (Lloyd et al., 1975). No bone sarcomas occurred among the 5 toxicity beagles injected with 253Es, excluding the one dog that subsequently received a large dose of 249Cf. This suggests that 20-day 253Es does not seem appreciably more toxic than the other transplutonium elements studied.
224Ra 1977-1979
Toxicity studies with 224Ra (quickradium, T1/2 = 3.62 days) were undertaken to understand the modifying effect of protraction on the dose- response of 224Ra observed in German patients (Spiess & Mays 1973; Mays et al., 1986). Four graded dose levels were administered over three injection spans. Groups 1-12 received their 224Ra in 50 weekly fractions to correspond to the average injection span in the German children; Groups 41-52 received a single injection, and Groups 81-92 received 10 weekly injections to correspond to the present treatment in Germany for ankylosing spondylitis. Most of the 224Ra given the beagles was prepared by the German Amersham-Buchler Firm which also prepares the 224Ra for the German patients. In a few instances, 224Ra of the same high radiochemical purity was prepared by Fred Bruenger in our Lab. The studies of 224Ra in beagles are among the most important with respect to understanding the mechanisms of alpha-particle-induced cancer. The short half-life of 224Ra causes some of it to decay on bone surfaces and some to decay within bone volume, giving a local distribution of dose in bone somewhat similar to that from 239pu. In the beagles receiving 278 rad from 224Ra protracted over 50 weeks (the only group in which all of the 224Ra dogs have died) the bone sarcoma appearance times and incidence were similar to that at the same skeletal dose from 239Pu. It remains to be seen, however, what the effectiveness of 224Ra will be at lower doses and shorter protraction times. The 224Ra study, being the most recent, has the largest number of living dogs.
TOXICITY STUDIES IN YOUNG AND OLD BEAGLES
Because of concern about the effects of radionuclides on members of the General Public with widely different ages, the studies in beagles were expanded to include administration at 3 months of age (to represent children) and 5 years of age (to represent middle-aged adults). 239Pu was selected as the bone surface seeker of greatest concern, while 226Ra was chosen to represent the bone volume seekers. Much attention has been given to the changing distribution of radioactivity in these dogs and to the observed biological effects.
Table 1 gives the status of each of the toxicity studies described in this “Guide to Beagle Studies at the University of Utah.” The test dogs are not included in this tabulation.
Most of the surviving dogs either received 224Ra as young adults or received 239Pu or 226Ra at 3 months of age. Optimum intercomparative information can be obtained if the protocol for the animals that have died continues to be followed for those still living.
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