Extracellular signaling through the microenvironment: a hypothesis relating carcinogenesis, bystander effects, and genomic instability.

Cell growth, differentiation and death are directed in large part by extracellular signaling through the interactions of cells with other cells and with the extracellular matrix; these interactions are in turn modulated by cytokines and growth factors, i.e. the microenvironment. Here we discuss the idea that extracellular signaling integrates multicellular damage responses that are important deterrents to the development of cancer through mechanisms that eliminate abnormal cells and inhibit neoplastic behavior. As an example, we discuss the action of transforming growth factor beta (TGFB1) as an extracellular sensor of damage. We propose that radiation-induced bystander effects and genomic instability are, respectively, positive and negative manifestations of this homeostatic process. Bystander effects exhibited predominantly after a low-dose or a nonhomogeneous radiation exposure are extracellular signaling pathways that modulate cellular repair and death programs. Persistent disruption of extracellular signaling after exposure to relatively high doses of ionizing radiation may lead to the accumulation of aberrant cells that are genomically unstable. Understanding radiation effects in terms of coordinated multicellular responses that affect decisions regarding the fate of a cell may necessitate re-evaluation of radiation dose and risk concepts and provide avenues for intervention.

Biomarkers of exposure and dose: state of the art.

Biomarkers provide methods to measure changes in biological systems and to relate them to environmental insults and disease processes. Biomarkers can be classified as markers of exposure and dose, markers of sensitivity, and markers of disease. It is important that the differences and applications of the various types of biomarkers be clearly understood. The military is primarily interested in early biomarkers of exposure and dose that do not require high levels of sensitivity but can be used to rapidly triage war fighters under combat or terrorist conditions and determine which, if any, require medical attention. Biomarkers of long-term radiation risk represent the second area of interest for the military. Biomarkers of risk require high sensitivity and specificity for the disease and insult but do not require rapid data turnaround. Biomarkers will help provide information for quick command decisions in the field, characterise long-term troop risks and identify early stages of radiation-induced diseases. This information provides major positive reassurances about individual exposures and risk that will minimise the physical and psychological impact of wartime radiation exposures.

Induction and repair of HZE induced cytogenetic damage.

Wistar rats were exposed to high-mass, high energy (HZE) 56Fe particles (1000 GeV/AMU) using the Alternating Gradient Synchrotron (AGS). The animals were sacrificed at 1-5 hours or after a 30-day recovery period. The frequency of micronuclei in the tracheal and the deep lung epithelial cells were evaluated. The relative effectiveness of 56Fe, for the induction of initial chromosome damage in the form of micronuclei, was compared to damage produced in the same biological system exposed to other types of high and low-LET radiation. It was demonstrated that for animals sacrificed at short times after exposure, the tracheal and lung epithelial cells, the 56Fe particles were 3.3 and 1.3 times as effective as 60Co in production of micronuclei, respectively. The effectiveness was also compared to that for exposure to inhaled radon. With this comparison, the 56Fe exposure of the tracheal epithelial cells and the lung epithelial cells were only 0.18 and 0.20 times as effective as radon in the production of the initial cytogenetic damage. It was suggested that the low relative effectiveness was related to potential for 'wasted energy' from the core of the 56Fe particles. When the animals were sacrificed after 30 days, the slopes of the dose-response relationships, which reflect the remaining level of damage, decreased by a factor of 10 for both the tracheal and lung epithelial cells. In both cases, the slope of the dose-response lines were no longer significantly different from zero, and the r2 values were very high. Lung epithelial cells, isolated from the animals sacrificed hours after exposure, were maintained in culture, and the micronuclei frequency evaluated after 4 and 6 subcultures. These cells were harvested at 24 and 36 days after the exposure. There was no dose-response detected in these cultures and no signs of genomic instability at either sample time.

Biomarkers of exposure, sensitivity and disease.

PURPOSE: This review is to evaluate the use of biomarkers as an indication of past exposure to radiation or other environmental insults, individual sensitivity and risk for the development of late occurring disease. OVERVIEW: Biomarkers can be subdivided depending on their applications. Markers of exposure and dose can be used to reconstruct and predict past accidental or occupational exposures when limited or no physical measurements were available. Markers of risk or susceptibility can help identify sensitivity individuals that are at increased risk for development of spontaneous disease and may help predict the increased risk in sensitive individuals associated with environmental or therapeutic radiation exposures. Markers of disease represent the initial cellular or molecular changes that occur during disease development. Each of these types of biomarkers serves a unique purpose. OUTLINE: This paper concentrates on biomarkers of dose and exposure and provides a brief review of biomarkers of sensitivity and disease. The review of biomarkers of dose and exposure will demonstrate the usefulness of biomarkers in evaluation of physical factors associated with radiation exposure, such as LET, doserate and dose distribution. It will also evaluate the use of biomarkers to establish relationships that exist between exposure parameters such as energy deposition, environmental concentration of radioactive materials, alpha traversals and dose. In addition, the importance of biological factors on the magnitude of the biomarker response will be reviewed. Some of the factors evaluated will be the influence of species, tissue, cell types and genetic background. The review will demonstrate that markers of sensitivity and disease often have little usefulness in dose-reconstruction and, by the same token, many markers of dose or exposure may not be applicable for prediction of sensitivity or risk.

Cellular effects of individual high-linear energy transfer particles and implications for tissue response at low doses.

The energy deposition patterns produced by the radiation environment in space can be quite different from those in conventional radiation environments. Furthermore, conventional radiation biological experiments, using randomly distributed particle tracks, cannot access some variables which may be important in determining the health effects of irradiation. Controlled microbeam irradiation provides the means to investigate the effects and unique energy deposition patterns and cell environment for a variety of end points.

Induction of micronuclei in respiratory tract following radon inhalation.

Male Wistar rats were exposed to radon and its progeny (0.0, 60, 262 and 564 working level months, WLM), and the frequency of micronuclei was determined in deep lung fibroblasts, and deep lung, trachea and nasal epithelial cells with slopes of 0.28, 0.67, 0.34 and 0.11 micronuclei/1000 binucleated cells/WLM respectively. Micronuclei in deep lung fibroblasts, isolated and cultured using two methods and media, demonstrated no differences in slopes. Biological damage was used as a biodosimeter to calculate the relationship between dosimetric units: alpha particle traversals or 'nuclear hits', dose in mGy and exposure in WLM. The estimated number of nuclear alpha traversals/Gy was 6.3. Radon exposure to 170 WLM resulted in the same frequency of micronuclei in deep lung epithelial cells as produced by one alpha hit/cell nucleus. Absorbed dose/unit of exposure (mGy/WLM) was estimated assuming the damage was related to absorbed dose or to changes in cell sensitivity and ranged from 1.13 to 1.34 for deep lung epithelial cells, 0.47 to 1.09 for deep lung fibroblasts, 0.34 to 0.67 for tracheal epithelial cells and 0.18 to 0.33 for nasal epithelial cells. Biological dosimetry can be used to relate exposure to damage, compare dosimetric units and validate physical dosimetry models. This approach can be applied to any inhaled material capable of producing biological damage.

Comparative clastogenic sensitivity of respiratory tract cells to gamma rays.

To understand the relationships between exposure and damage to different cell populations in the respiratory tract, methods were developed to culture deep-lung fibroblasts and epithelial cells from the nose, trachea and deep lungs. Female F-344 Fischer and male Wistar rats were exposed to 1-5 Gy of 60Co gamma rays at a dose rate of 0.4 Gy/min. Cells were isolated for short-term culture, and the incidences of binucleated cells and micronuclei were determined. The incidences of micronuclei were determined in cytochalasin-B-induced binucleated cells at 72 h for nasal and tracheal tissue and 96 h for deep-lung fibroblasts and epithelial cells. Maximum frequencies of binucleated cells were found in the control nonirradiated cells at these harvest times, and the frequencies were not significantly affected at these harvest times by radiation exposure. No significant differences were found in the frequencies of micronuclei induced in the nasal epithelial cells isolated from female F-344 Fischer or male Wistar rats. Fibroblasts cultured in different media and isolated from either female F-344 Fischer or male Wistar rats also showed a similar frequency of micronuclei. Over the doses tested, the frequency of micronuclei in the respiratory tract cells increased linearly with the dose. The slopes were 92.2 +/- 9.2, 76.2 +/- 7.9, 32.8 +/- 2.4 and 28.7 +/- 3.4 micronuclei/1000 binucleated cells/Gy for deep-lung epithelial cells, deep-lung fibroblasts, tracheal epithelial cells and nasal epithelial cells, respectively. Deep-lung epithelial or fibroblast cells were about two to three times as sensitive for elastogenic damage as nasal and tracheal epithelial cells. The measurement of micronuclei in isolated respiratory tract cells is very useful in assessing cytogenetic damage induced in different cell types by radiation.

Lung cancer response following inhaled radon in the A/J and C57BL/6J mouse.

A/J and C57BL/6J mice, strains which differ in susceptibility to chemical-induced lung tumours, were examined for differential sensitivity to radon-induced lung cancer. Sixty weeks after exposure to 952 WLM radon (3.34 J h1 m-3), no lung tumours were found in the control, sham- (one dust-) exposed, or radon-exposed C57BL/6J mice. In contrast, each of the A/J groups had a substantial number of tumours. Although there were no differences in overall lung tumour incidence or multiplicity associated with radon and ore dust exposure in A/J mice, distinct differences in tumour size and histopathological appearance were noted. The ratio of lung adenoma-to-carcinoma found in control A/J mice was 10:1, whereas the ratio in one dust-exposed animals was 10:8 and that in radon-exposed animals was 8:6. Based on these data, particulates appear to modify the promotion-progression of lung cancer in this strain. In addition, the appearance of two very large carcinomas histologically resembling Clara-cell-derived tumours only in the radon-exposed A/J mice and the possibility of alpha-particle-induced killing of target cells in the radon-exposed animals suggests that A/J but not the C57BL/6J, mice may have genes that modify susceptibility to radon-induced lung cancer.

The biological effectiveness of radon-progeny alpha particles. V. Comparison of oncogenic transformation by accelerator-produced monoenergetic alpha particles and by polyenergetic alpha particles from radon progeny.

Generation of estimates of risk caused by exposure to radon in the home, either from miner data or from A-bomb data, requires several scaling factors such as for dose, dose rate and radiation quality, and possible synergisms. Such scaling factors are best developed from laboratory-based studies. Two possible sources of alpha particles for such studies are (1) a polyenergetic spectrum, generated directly by radon and its progeny, or (2) a series of monoenergetic alpha particles. We compare here the results of oncogenic transformation from studies using both systems. At the Columbia University Radiological Research Accelerator Facility (RARAF), C3H 10T1/2 cells were irradiated with alpha particles of various energies, with defined LETs from 70 to 200 keV/mum. At Pacific Northwest Laboratory, cells from the same stock were exposed to alpha particles from radon gas and its progeny, which were in equilibrium with the culture medium. There was good agreement between the results of oncogenic transformation experiments using the two different exposure systems. Apart from the experimental transformation frequencies themselves, such a comparison requires (1) reliable dosimetry at both facilities and (2) estimated LET distributions for the polyenergetic alpha-particle irradiator. Thus this good agreement gives some confirmation to the technique which is used to fold together oncogenic transformation rates from monoenergetic alpha particles to yield a predicted rate for a spectrum of alpha particles.

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.

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)

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)

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.

Pulmonary toxicity of inhaled diesel exhaust and carbon black in chronically exposed rats. Part I: Neoplastic and nonneoplastic lung lesions.

This study compared the pulmonary carcinogenicities and selected noncancer effects produced by chronic exposure of rats at high rates to diesel exhaust and carbon black. The comparison was intended to provide insight into the likely importance of the mutagenic organic compounds associated with the soot portion of diesel exhaust in inducing pulmonary carcinogenicity in diesel exhaust-exposed rats. The role of the organic fraction has become important in judging the usefulness of the substantial data base on carcinogenicity in rats for predicting lung cancer risk for humans, and for determining the most appropriate method of extrapolating results across species and exposure concentrations. Rats were exposed chronically to either diesel exhaust or carbon black, which served as a surrogate for diesel exhaust soot with much reduced mutagenic activity associated with its organic fraction. The sequestration of particles in the lung and the induction of pulmonary neoplasia and non-neoplastic changes in the lung were compared in detail. Samples also were provided to collaborators to examine adduct formation in lung DNA and hemoglobin. Approximately 140 female and 140 male F344/N rats were exposed for 16 hours per day, 5 days per week for up to 24 months, beginning at eight weeks of age, to diesel exhaust or carbon black at 2.5 mg or at 6.5 mg particles/m3 of air, or to clean air as controls. The diesel exhaust was generated by light-duty engines burning certification fuel and operating on an urban-duty cycle. The carbon black was selected because it had particle size and surface area characteristics similar to those of diesel exhaust soot, but markedly less mutagenic activity associated with its organic fraction when analyzed using procedures typically used in studies of diesel soot. Rats were killed after 3, 6, 12, 18, or 23 months of exposure to measure lung and lung-associated lymph node burdens of particles, lung weight, bronchoalveolar lavage indicators of inflammation, DNA adducts in whole lung and alveolar type II cells, and chromosome injury in circulating lymphocytes, and to perform histopathologic assessment. In addition, after 3 and 18 months of chronic exposure, one group of rats was acutely exposed to radiolabeled carbon black particles or to fluorescent microspheres. These exposures were conducted to examine the clearance of radiolabeled particles and the sequestration of the fluorescent microspheres in the lungs. These experiments provided information on clearance overload and particle dosimetry. The growth characteristics of lung neoplasms also were examined by transplanting neoplastic cells into athymic mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Micronuclei induced by radon and its progeny in deep-lung fibroblasts of rats in vivo and in vitro.

Genotoxic damage induced by radon and its progeny was investigated using the micronucleus assay in deep-lung fibroblasts to compare the response induced in vitro with that induced from inhalation of radon and its progeny in vivo. Male Wistar rats were exposed to 0, 115, 213 and 323 working-level months (WLM) of radon and its progeny by inhalation. After sacrifice, the cells were isolated and grown in culture, and the frequency of micronuclei was determined. A linear increase in the frequency of micronuclei was measured as a function of exposure [micronuclei/1000 binucleated cells = (29 +/- 9) + (0.47 +/- 0.04) WLM]. To compare exposure in WLM to dose in mGy, and to study how cell proliferation influences the way inhalation of radon and its progeny induces micronuclei, lung fibroblasts were isolated and exposed in vitro to graded doses from radon and its progeny after either 16 or 96 h in tissue culture. Cell cycle stage at the time of exposure was determined using flow cytometry. Primary lung fibroblasts exposed as either nondividing or dividing cells showed dose-dependent increases in micronuclei [micronuclei/1000 binucleated cells = (33 +/- 40) + (593 +/- 68)D and micronuclei/1000 binucleated cells = (27 +/- 69) + (757 +/- 88)D, respectively, where D is dose in Gy]. Results showed no significant influence (P = 0.20) of cell proliferation at the time of exposure on the frequency of micronuclei induced by radon and its progeny. Comparing dose-response relationships for nondividing cells to the exposure response for cells exposed by inhalation of radon and its progeny, it was estimated that a 1-WLM exposure in vivo caused the same amount of cytogenetic damage as produced by 0.79 mGy in vitro. In vivo/in vitro research using the micronucleus assay in lung fibroblasts serves as a powerful tool to estimate effective dose to cells in the respiratory tract after inhalation of radon and its progeny. Such studies form the basis for understanding the relationship between exposure, dose and biological damage.

Metaphase chromosome aberrations as markers of radiation exposure and dose.

Chromosome aberration frequency provides the most reliable biological marker of dose to detect acute accidental radiation exposure. Significant radiation-induced changes in the frequency of chromosome aberrations can be detected at very low doses (Lloyd et al., 1992). In animal studies chromosome aberrations provide a method to relate exposure to cellular dose. Using an in vivo/in vitro approach, aberrations provided a biological marker of dose from radon progeny exposure, which was used to convert exposure, work level months (WLM) to dose in grays (Gy) delivered to rat tracheal epithelial cells. Injection of Chinese hamsters with 144Ce, which produced a low-dose rate exposure of bone marrow to low-linear energy transfer (LET) radiation, increased the cell sensitivity for the induction of chromatid exchanges by subsequent external 60Co exposure. Our paper provides information on using molecular chromosome probes to "paint" chromosomes and score chromosome damage. This approach illustrates how technical advances make it possible to understand the mechanisms involved in the formation of chromosome aberrations. These studies demonstrate the usefulness of chromosome damage as a biological marker of dose and cellular responsiveness.

Distribution and biological effects of inhaled 239Pu(NO3)4 in cynomolgus monkeys.

Twenty male cynomolgus monkeys were exposed by inhalation either to an aerosol of 239Pu(NO3)4 to produce projected initial lung burdens of either 40, 10, or 4 kBq or to a carrier aerosol as a control. Animals died or were sacrificed at 0.01, 1, 3, 6, 12, 24, 40, and 99 months after inhalation, and the distribution and biological effects of the 239Pu were determined. The 239Pu cleared efficiently from the lungs so that less than 0.05 kBq remained at 99 months after exposure to 40 kBq. Total skeletal 239Pu activity was nearly constant after the first year, but the fraction of the body burden in skeleton at sacrifice increased with time up to 99 months because of clearance from other organs. Plutonium in the liver increased to a peak at 1 year and then decreased to about 10% of the peak value at 99 months. Plutonium in the testes was localized in the interstitial tissue with only 0.01 to 0.002% of the projected lung burden remaining in testes at 99 months after inhalation. Three animals exposed to 40 kBq of 239Pu died of radiation-related pulmonary pneumonitis and fibrosis. A primary papillary adenocarcinoma of the lung was identified in one animal exposed to 40 kBq initial lung burden and sacrificed 99 months after inhalation. The frequency of chromosome aberrations in blood lymphocytes was significantly elevated only in monkeys with projected deposits of 40 kBq of 239Pu. There was no change in aberration frequency in other exposure groups as a function of inhaled activity, time after exposure, or calculated total dose to the lungs. Only in monkeys that had marked radiation-induced pathological changes in the lung did the frequency of chromosome-type aberrations increase significantly, to a value about twice the control level. In cynomolgus monkeys, chromosome aberration frequency in blood lymphocytes is not a good indicator of radiation dose or damage from inhaled soluble plutonium.

Changes in Activities of Enzymes of Carbon Metabolism in Leaves during Exposure of Plants to Low Temperature.

The aim of this study was to determine the response of photosynthetic carbon metabolism in spinach and bean to low temperature. (a) Exposure of warm-grown spinach and bean plants to 10 degrees C for 10 days resulted in increases in the total activities of a number of enzymes, including ribulose 1,5-bisphosphate carboxylase (Rubisco), stromal fructose 1,6 bisphosphatase (Fru 1,6-P(2)ase), sedoheptulose 1,7-bisphosphatase (Sed 1,7-P(2)ase), and the cytosolic Fru 1,6-P(2)ase. In spinach, but not bean, there was an increase in the total activity of sucrose-phosphate synthase. (b) The CO(2)-saturated rates of photosynthesis for the cold-acclimated spinach plants were 68% greater at 10 degrees C than those for warm-acclimated plants, whereas in bean, rates of photosynthesis at 10 degrees C were very low after exposure to low temperature. (c) When spinach leaf discs were transferred from 27 to 10 degrees C, the stromal Fru 1,6-P(2)ase and NADP-malate dehydrogenase were almost fully activated within 8 minutes, and Rubisco reached 90% of full activation within 15 minutes of transfer. An initial restriction of Calvin cycle fluxes was evident as an increase in the amounts of ribulose 1,5-bisphosphate, glycerate-3-phosphate, Fru 1,6-P(2), and Sed 1,7-P(2). In bean, activation of stromal Fru 1,6-P(2)ase was weak, whereas the activation state of Rubisco decreased during the first few minutes after transfer to low temperature. However, NADP-malate dehydrogenase became almost fully activated, showing that no loss of the capacity for reductive activation occurred. (d) Temperature compensation in spinach evidently involves increases in the capacities of a range of enzymes, achieved in the short term by an increase in activation state, whereas long-term acclimation is achieved by an increase in the maximum activities of enzymes. The inability of bean to activate fully certain Calvin cycle enzymes and sucrose-phosphate synthase, or to increase nonphotochemical quenching of chlorophyll fluorescence at 10 degrees C, may be factors contributing to its poor performance at low temperature.