The role of dose rate in the induction of micronuclei in deep-lung fibroblasts in vivo after exposure to cobalt-60 gamma rays.

To evaluate the influence of low-dose-rate exposures on biological damage, it is necessary to have cells that can be maintained in the same stage of the cell cycle for long periods. Normal rat lung fibroblasts represent a stable cell type with a slow turnover rate in vivo. These cells can be stimulated to divide by placing them in tissue culture. Therefore, a constant cell population can be exposed over a protracted time and stimulated to divide, and the cytogenetic damage can be evaluated at the first cell division after exposure. By placing rats at different distances from a 60Co source, they were exposed to graded doses of gamma rays--0.0, 3.9, 7.4 and 11.3 Gy--protracted over either 4 or 67 h. Fibroblasts were isolated from the lung and cultured for 24 h; after cytochalasin B was added, the cells were cultured for an additional 69 to 72 h. The percentage binucleated cells in fibroblasts of animals exposed for 4 or 67 h was 47.1 +/- 4.3 and 62.1 +/- 3.9. There was no influence of dose on the percentage binucleated cells, but the fraction of cells that divided at 67 h was significantly higher (P < 0.05) than observed at 4 h. Cells were scored for micronuclei on coded slides. The dose-response data from animals exposed for 4 and 67 h were fitted to the following linear dose-response relationships, where D = dose; micronuclei/binucleated cell = 0.02 +/- 0.03 + 2.38 +/- 0.44 x 10(-2) D, and micronuclei/binucleated cell = 0.01 +/- 0.06 + 1.01 +/- 0.10 x 10(-2) D, respectively. The r2 values for the two curves were 0.67 and 0.91, indicating the goodness of fit for the data for the 4- and 67-h treatments. The slopes were different from zero and each other at the P < 0.05 level of significance. The effectiveness of the 60Co exposure decreased as the dose rate decreased. At dose rates below 0.17 Gy/h, the effectiveness remained constant over the range of doses and dose rates used. Comparing the slope of the dose response for the lowest exposure rate to that from information published previously, the dose-rate effectiveness factor was 6.14 +/- 0.65 for the induction of micronuclei in deep-lung fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

Clastogenic effects of defined numbers of 3.2 MeV alpha particles on individual CHO-K1 cells.

Research to determine the effects of defined numbers of alpha particles on individual mammalian cells is helpful in understanding risks associated with exposure to radon. This paper reports the first biological data generated using the single-particle/single-cell irradiation system developed at Pacific Northwest Laboratory. Using this apparatus, CHO-K1 cells were exposed to controlled numbers of 3.2 MeV alpha particles, and biological responses of individual cells to these irradiations were quantified. Chromosomal damage, measured by the induction of micronuclei, was evaluated after no, one, two, three or five particle traversals. Exposures of up to five alpha particles had no influence on the total numbers of cells recovered for scoring. With increased numbers of alpha particles there was a decrease in the ratio of binucleated to mononucleated cells of 3.5%/hit, suggesting that alpha particles induced dose-dependent mitotic delay. A linear hit-response relationship was observed for micronucleus induction: Micronuclei/binucleated cell = 0.013 +/- 0.036 + (0.08 +/- 0.013) x D, where D is the number of particles. When the estimated dose per alpha-particle traversal was related to the frequency of induced micronuclei, the amount of chromosomal damage per unit dose was found to be similar to that resulting from exposures to alpha particles from other types of sources. Approximately 72% of the cells exposed to five alpha particles yield no micronuclei, suggesting the potential for differential sensitivity in the cell population. Additional studies are needed to control biological variables such as stage of the cell cycle and physical parameters to ensure that each cell scored received the same number of nuclear traversals.

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.

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.

Susceptibility of owl monkeys to Plasmodium falciparum in relation to hemoglobin and karyotype.

Humans with inherited abnormalities of hemoglobin (Hb) synthesis have less frequent and less severe infections of malaria. This study sought to determine if karyotypic variation in the owl monkey was expressed as differences in Hb moieties and if it offered a selective advantage in susceptibility to malaria. Five karyotypes of owl monkey were evaluated on the basis of the electrophoretic mobility of their major and minor Hb components. The results of 40 owl monkeys of different karyotypes demonstrated that statistically significant differences exist among karyotype I animals and those with karyotypes II, III, and V, particularly with regard to their HbA2 concentrations. This finding is of interest in light of the fact that karyotype I animals are considered to be less susceptible to infection with human strains of Plasmodium falciparum than karyotypes II, III, and V, which are viewed as being highly susceptible.

Epidermal carcinogenesis studies of synthetic fossil fuel materials in mice.

Skin tumor response in mice to solvent fractions of heavy distillate (HD) from a solvent-refined coal (SRC-II) process indicated that the basic tar and neutral tar were the most carcinogenically potent fractions. Assays of another SRC-II coal liquid that had been fractionally distilled indicated that the carcinogenicity of this material for mouse skin is due to that portion boiling above 371 degrees C (700 degrees F), and that the carcinogenic potency of the material increased with boiling point. Samples of the 399-427 degrees C (750-800 degrees F) distillate were nitrosated to destroy primary aromatic amines and were chemically fractionated to assess the carcinogenicity of chemical class fractions of these complex mixtures. Data from these assays indicated that neutral polycyclic aromatic hydrocarbons (PAH) and nitrogen-containing polycyclic aromatic compounds (NPAC) both contribute to the carcinogenicity of this distillate.

Reduction of the nocturnal rise in pineal melatonin levels in rats exposed to 60-Hz electric fields in utero and for 23 days after birth.

Rats exposed to 60-Hz electric fields of either 10, 65, or 130 kV/m from conception to 23 days of age exhibited reduced peak nighttime pineal melatonin contents compared to unexposed controls. As a group, the exposed rats also exhibited a phase delay, estimated at approximately 1.4 hours, in the occurrence of the nocturnal melatonin peak. No clear dose-response relationship was noticed over the range of electric field strengths used as treatments in these experiments. These are the first studies concerned with the effects of electric field exposure on the pineal melatonin rhythm in immature rats. The findings are generally consistent with those obtained using adult rats, where electric field exposure has been shown to abolish the nighttime rhythm in pineal melatonin concentrations.

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.

Effectiveness of radon relative to acute 60Co gamma-rays for induction of micronuclei in vitro and in vivo.

Because radon and its progeny (referred to collectively here as radon) emit alpha particles with a wide range of energies, as well as beta particles and gamma-rays, it is important to quantitate the relationship between initial damage induced by radon and that by acute low-LET radiation. We have evaluated dose-response relationships for induction of micronuclei both in vivo and in vitro following exposure to radon or 60Co. To determine if isolation procedures altered the cells' responsiveness to 60Co gamma-ray exposures, animals were exposed before cell isolation, or cells were isolated and then exposed. The data were described by linear dose-response functions and were not significantly different when the radiation exposure was in vivo or in vitro (respectively micronuclei/1000 binucleated cells = 1.6 +/- 6.5 + 62 +/- 2.7 D; micronuclei/1000 binucleated cells = 15.4 +/- 26.0 + 54.6 +/- 11.4 D, where D is in Gy). Primary rat lung fibroblasts (RLF) or Chinese hamster ovary (CHO-K1) cells were exposed in vitro to either radon or 60Co gamma-rays. Radon was 10.9 +/- 2.6 and 12.5 +/- 2.4 times as effective per Gy of radiation dose in producing micronuclei as was 60Co in RLF and CHO-K1 cells respectively. To determine the relative biological effectiveness of in vivo radon exposure, animals were exposed to either radon or 60Co, and lung fibroblasts were isolated and evaluated for radiation-induced micronuclei. In vivo radon exposure was 10.6 +/- 1.0 times as effective as acute whole-body 60Co exposure in producing micronuclei in lung fibroblasts. Different cell lines and exposure conditions resulted in similar effectiveness factors. Such ratios help evaluate the biological damage, hazard and risk associated with radon inhalation.

Inhaled radon-induced genotoxicity in Wistar rat, Syrian hamster, and Chinese hamster deep-lung fibroblasts in vivo.

This study was performed (1) to provide a comparison of the genotoxic effects of inhaled radon and radon progeny, referred to as radon in this paper, among three species of rodents: Wistar rats, Syrian hamsters, and Chinese hamsters; (2) to determine if initial chromosome damage was related to the risk of induction of lung cancer; and (3) to evaluate the tissue repair and long-term presence of cytogenetic damage in respiratory tract cells. These species were selected because Syrian hamsters are very resistant to radon induction of lung cancer and Wistar rats are sensitive; no literature is available on the in vivo effects of radon in the Chinese hamster. Exposure-response relationships were established for the rats and Syrian hamsters while the Chinese hamsters received a single exposure of radon. At 4 h (0.2 days), 15 days, and 30 days after the highest WLM exposure to radon, Wistar rats, Chinese hamsters, and Syrian hamsters were killed, and lung fibroblasts were isolated and grown in culture to determine the frequency of induced micronuclei. Animals at each level of exposure showed an increase in the frequency of micronuclei relative to that in controls (P < 0.05). The exposure-response relationship data for rats and Syrian hamsters killed 0.2 days after the end of exposure were fit to linear equations (micronuclei/1000 binucleated cells = 15.5 +/- 14.4 + 0.53 +/- 0.06 WLM and 38.3 +/- 15.1 + 0.80 +/- 0.08 WLM, respectively). For the single exposure level used (496 WLM) in Chinese hamsters killed at 0.2 days after exposure, the frequency of micronuclei/1000 binucleated cells/WLM was 1.83 +/- 0.02. A comparison of the sensitivity for induction of micronuclei/WLM illustrated that Chinese hamsters were three times more sensitive than rats. The Syrian hamsters also showed a significantly elevated response (P < 0.05) relative to rats. These data suggest that initial chromosome damage is not the major factor responsible for the high rate of radon-induced cancer in rats relative to Syrian hamsters. The frequency of micronuclei in radon-exposed rats, Syrian hamsters, and Chinese hamsters significantly decreased (P < 0.05) as a function of time after the exposure. The rate of loss of damaged cells from the lung was greatest in the Chinese hamsters, followed by Wistar rats and Syrian hamsters, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Comet assay reveals DNA strand breaks induced by ultrasonic cavitation in vitro.

The induction of DNA strand breaks in cultured Chinese hamster ovary (CHO) cells was investigated in suspensions directly exposed to 2.17-MHz ultrasound. Production of hydrogen peroxide, a DNA-damaging sonochemical, by inertial cavitation was enhanced by the use of argon-and-oxygen-bubbled media and cell survival was improved by establishing standing waves and minimizing tube rotation. Viable cells were separated from the suspension after exposure and kept on ice for evaluation with the single-cell gel electrophoresis (comet) assay. With this assay, DNA damage from as little as 2-mumol/L hydrogen peroxide treatment for 30 min could be detected, and cell survival as low as 2-5% after ultrasound exposure was adequate for assay. An ultrasound dose-response trend was noted for increasing pressure amplitude up to 0.82 MPa (free field) and increasing exposure duration up to 4 min. The cells were able to repair some of the strand breaks when warmed to 28 degrees C for 30 min. The effect was not eliminated by addition of catalase, which indicates that the DNA damage was not due to the action of residual H2O2 alone. The results confirm the hypothesis of DNA damage in cells surviving inertial cavitation.

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.

Experience with a routine fecal sampling program for plutonium workers.

A quarterly fecal sampling program was conducted at the U. S. Department of Energy's Hanford site for congruent to 100 workers at risk for an intake of plutonium oxide and other forms of plutonium. To our surprise, we discovered that essentially all of the workers were excreting detectable activities of plutonium. Further investigation showed that the source was frequent, intermittent intakes at levels below detectability by normal workplace monitoring, indicating the extraordinary sensitivity of fecal sampling. However, the experience of this study also indicated that the increased sensitivity of routine fecal sampling relative to more common bioassay methods is offset by many problems. These include poor worker cooperation; difficulty in distinguishing low-level chronic intakes from a more significant, acute intake; difficulty in eliminating interference from ingested plutonium; and difficulty in interpreting what a single void means in terms of 24-h excretion. Recommendations for a routine fecal program include providing good communication to workers and management about reasons and logistics of fecal sampling prior to starting, using annual (instead of quarterly) fecal sampling for class Y plutonium, collecting samples after workers have been away from plutonium exposure for a least 3 d, and giving serious consideration to improving urinalysis sensitivity rather than going to routine fecal sampling.

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.

Neptunium-237 inhalation in rats.

Groups of rats were exposed to aerosols of 237Np nitrate to determine clearance rates, retention and distribution at various intervals after inhalation. Initial lung burdens (ILB) after 237Np inhalation by three treatment groups were 0.12, 0.19 and 0.37 mu Ci/kg, respectively. Radiochemical analyses of animals killed at 4, 8, 14, 28 and 90 d, as well as data for others maintained until they became moribund, showed that their lung clearance followed a three-compartment model, clearance half-times for which were 1, 35, and 10,000 d, respectively. Only 3% of the ILB was retained after 90 d; 12% of that burden had translocated to the skeleton at 750 d; the half-time for skeletal retention was 2500 d. A single tumor was the only malignancy detected in the lungs of the 35 animals allowed to survive the early phase of the study.

The distribution and effects of inhaled 239Pu(NO3)4 deposited in the liver of dogs.

The distribution and effects of inhaled 239Pu(NO3)4 deposited in the liver of dogs were studied in five groups of 20 beagles exposed to initial lung depositions ranging from 1.0 to 520 Bq g(-1) lung. Following life-span observations, the liver contained 40 +/- 1% of the final body deposition of plutonium, second only to the skeleton. The liver-to-skeleton ratio of deposited plutonium for total organ was 0.8, or 3.5 when expressed on a per-gram basis. There was no effect of exposure level on liver-to-skeleton ratios. Autoradiographs showed that the dose rate delivered to parenchymal cells was higher than evident from radiochemical analysis of the whole organ. Elevated levels of serum liver enzymes were observed in groups with mean liver concentrations of 1.3 Bq g(-1) and liver doses of 3 Gy or higher. Nodular hyperplasia of liver and bile-duct hyperplasia were observed. Liver tumors, principally of bile-duct epithelium, were late-occurring and were observed at lower exposure levels at which life span was not shortened by lung or bone tumors.

A new method for analyzing high-resolution spectra from whole-body counter in-vivo measurements.

A new method has been developed at the Pacific Northwest Laboratory to analyze pulse-height energy spectra from whole-body counter in-vivo examinations that use high-resolution Ge detectors. A simple data transformation and smoothing function is used to calculate background and identify photopeaks for isotopic analysis. This technique is beneficial for routine in-vivo measurement programs because it avoids dependence upon complex spectrum deconvolution, stripping, or other least-squares fitting techniques that complicate the assessment of measurement reliability. An in-vivo measurement spectrum is analyzed by first applying the variance stabilizing transformation to the data in each channel, which results in a variable with unit variance. A background spectrum is then determined by smoothing the transformed data. Finally, peaks are identified whenever the difference between the background spectrum and the transformed measurement spectrum exceeds 2.57 standard deviations. This method of spectrum analysis is especially suited to whole-body counting because background spectra are calculated as an integral part of the analysis procedure. This new technique has been applied successfully to routine in-vivo measurements of Pu, Am, and U. The computations for this new pulse height-energy spectrum analysis procedure are easily programmed on a desk-top or analyzer-based computer. The simplicity of the computational technique is also attractive because of the ease with which results can be verified.

Toxicology of 85Kr: effects of whole-body immersion exposure on newborn rats.

Newborn rats exposed to 85Kr exhibited acute radiation effects, e.g. epilation, skin scaling and abnormal development of the extremities, at beta immersion doses in excess of 1000 rad to the skin surface. The incidence of skin tumors, principally basal-cell carcinomas, was increased at all dose levels over the range from 1000 to 4750 rad. The effective skin-surface dose to induce basal-cell carcinoma in the newborn Wistar rat is apparently less than 1000 rad, the lowest dose employed in this study. No lung tumors attributable to 85Kr exposure were observed in these rats.