Long-term changes in mouse lung following inhalation of a fibrosis-inducing dose of 239PuO2: changes in collagen synthesis and degradation rates.

Mice were exposed by nose-only inhalation to 239PuO2, which resulted in an IAD of 1110 +/- 29 Bq. At various times after exposure, rates of collagen metabolism were measured using validated in vivo methods based on the administration of radiolabelled proline, together with a large flooding dose of unlabelled proline and measurement of its incorporation into lung collagen as hydroxyproline. Dramatic increases in both synthesis and degradation rates of collagen were observed. At 54 days after exposure the fractional synthesis rates in experimental mice were almost five times those in controls (control: 3.2 +/- 0.6%/day, 239PuO2-exposed: 14.5 +/- 0.4%/day) and by 300 days synthesis rates, although declining, were still more than double the control values. A similar pattern of change was observed for collagen degradation. The combination of changes in synthesis and degradation rates led to a 60% increase in lung collagen content by 300 days (control: 3.05 +/- 0.24 mg/lung, 239PuO2-exposed: 4.88 +/- 0.42 mg/lung). The data suggest that extensive remodelling of the lung connective tissue matrix occurs during development of fibrosis and that, over long periods of time, small imbalances between synthesis and degradation may result in quite large increases in protein content.

Effect of inhaled alpha-emitting nuclides on mouse alveolar macrophages.

The effects of inhaled alpha emitters on the free cell population of the mouse lung were investigated up to 100 days after exposure. Groups of mice inhaled aerosols of 238PuO2, 239PuO2, or 241Am(NO3)3 to give alveolar deposits resulting in lung-averaged cumulative absorbed doses of about 20 Gy by the end of the study. Initially, with 238Pu most of the activity was associated with relatively few pulmonary alveolar macrophages (PAM), whereas with 241Am, all pulmonary alveolar macrophages were labeled and a substantial fraction was extracellular. The free cell population of the lung was sampled using bronchoalveolar lavage. The main parameters investigated were (a) the recovery and total numbers of free cells, including PAM, lymphocytes, and neutrophils; (b) the incidence of nuclear abnormalities in PAM (cells with more than one nucleus or with micronuclei); and (c) metabolic activation of PAM from measurements of their size and associated beta-glucuronidase activity. All three actinides produced depletions in total numbers of PAM, increased incidences of nuclear abnormalities, and metabolic activation of PAM, without a marked infiltration of inflammatory cells. Americium-241, which is distributed relatively uniformly in PAM, produced the most marked changes in that population and 238Pu, which gave the most inhomogeneous distribution of activity, produced the least.

The effect of firing temperature on the lung retention and translocation of Pu following the inhalation of 238PuO2 and 239PuO2 by CBA/H mice.

Mice were exposed by inhalation to sized aerosols of 238PuO2 and 239PuO2 which had been fired at temperatures from 550-1250 degrees C and groups killed at times between 1 d and 2 y after exposure. Measurements were made of 238Pu and 239Pu in the lungs, lung-associated lymph nodes, liver and skeleton. With 239Pu, lung retention and translocation were independent of firing temperature. With 238Pu on the other hand, the retention in lung was greater initially than for 239Pu but, with the low-fired oxide, eventually fell below that of 239Pu. With high-fired oxides, the lung retention of 238Pu still exceeded that of 239Pu after 2 y. Translocation to liver and bone was invariably greater for 238Pu than for 239Pu and was also dependent on firing temperature. The practical implications of these findings are discussed.

Preliminary studies of the interaction between 239PuO2 and cigarette smoke in the mouse lung.

Our current experiments were designed to show whether 12 months' exposure to cigarette smoke enhances the incidence of lung tumours in mice that had previously inhaled 239PuO2. These periods of smoke exposure are almost complete. After death their lungs will be cleared and any nodules found will be sectioned for histopathology. This paper reports the results of two preliminary experiments conducted earlier. The first study showed that mice could tolerate the proposed smoking regime for 3 months, with no sign of ill health in any animal throughout. The major difference found was a reduced growth rate in both smoke- and sham-exposed mice relative to that of cage controls. After 3 months of treatment, histopathology and morphometry of lung sections found only slight smoke-induced changes. These included a reduced proportion of alveolar space and an increased number of pulmonary alveolar macrophages (PAM) per unit area. Bronchopulmonary lavage showed that the PAM from smoke-exposed mice were larger than those from sham-exposed or control mice and that an increased proportion of cells were binucleate. All mice in the second study were initially exposed to 239PuO2, then subsequently divided into three treatment groups as above. Cigarette smoke exposure was shown to inhibit the removal of 239Pu from the lung whilst sham exposure had no effect. Smoke exposure also produced an increase and sham exposure a decrease in lung weights relative to those of cage controls. The latter was probably as a result of their lower growth rate. In our current experiments it is likely that the group receiving 239PuO2, then smoke, will receive a higher radiation dose to lung than those receiving 239PuO2 only. Any increased tumour incidence found will be considered in conjunction with this evidence.

Radiation effects in the lung.

This article outlines the principles of radiobiology that can explain the time of onset, duration, and severity of the complex reactions of the lung to ionizing radiation. These reactions have been assayed biochemically, cell kinetically, physiologically, and pathologically. Clinical and experimental data are used to describe the acute and late reactions of the lung to both external and internal radiation including pneumonitis, fibrosis and carcinogenesis. Acute radiation pneumonitis, which can be fatal, develops in both humans and animals within 6 months of exposure to doses greater than or equal to 8 Gy of low LET radiation. It is divisible into a latent period lasting up to 4 weeks; an exudative phase (3-8 weeks) and with an acute pneumonitic phase between 2 and 6 months. The latter is an inflammatory reaction with intra-alveolar and septal edema accompanied by epithelial and endothelial desquamation. The critical role of type II pneumonocytes is discussed. One favored hypothesis suggests that the primary response of the lung is an increase in microvascular permeability. The plasma proteins overwhelm the lymphatic and other drainage mechanisms and this elicits the secondary response of type II cell hyperplasia. This, in its turn, produces an excess of surfactant that ultimately causes the fall in compliance, abnormal gas exchange values, and even respiratory failure. The inflammatory early reaction may progress to chronic fibrosis. There is much evidence to suggest that pneumonitis is an epithelial reaction and some evidence to suggest that this early damage may not be predictive of late fibrosis. However, despite detailed work on collagen metabolism, the pathogenesis of radiation fibrosis remains unknown. The data on radiation-induced pulmonary cancer, both in man and experimental animals from both external and internal irradiation following the inhalation of both soluble and insoluble alpha and beta emitting radionuclides are reviewed. Emphasis is placed on the data showing that alpha emitters are at least an order of magnitude more hazardous than beta/gamma radiation and on recent data showing that the more homogeneous the irradiation of the lung, the greater is the carcinogenic hazard which contradicts the so-called "hot particle" theory.

Translocation of 239Pu in mice following inhalation of sized 239PuO2.

This study showed that when SAS/4 mice were exposed to sized 239PuO2 only about 0.5% of the 239Pu was translocated from lung to other organs. The fraction of 239Pu translocated appeared to be independent of the particle size of the administered 239PuO2 within the range of AMADs investigated (0.8-2.2 microns). The distribution of translocated 239Pu was similar to that observed with other species in that most of the activity was associated with the lung-associated lymph nodes followed by bone and liver. The fraction of 239Pu translocated was comparable to that found in studies with rats (ICRP72) but less than that found in more extended studies with beagle dogs.

Macrophage depletion of mouse lung following inhalation of 239PuO2.

Changes in the free-cell population of the lungs of two strains of mice (SAS/4 and CBA/H) were studied up to 4 months after inhalation exposure to a sized fraction of 239PuO2 particles (1.5 micron AMAD) to give initial alveolar depositions (IADs) ranging from 17 to 810 Bq. A sample of the free-cell population of the lung was recovered by bronchoalveolar lavage, and a radiometric method was used to estimate the total number of pulmonary alveolar macrophages (PAM) in the lung. The response of the lung to 239PuO2 was characterized by an initial, dose-dependent depression in the total number of PAM following an IAD as low as 50 Bq. At IADs greater than 150 Bq, the initial depression continued for longer, merging into a chronic phase in which the PAM were larger and were accompanied by a minor infiltration of leukocytes. These findings were confirmed by histology, which also revealed focal accumulations of Type II pneumocytes. The results indicate that inhaled alpha-emitting particles are effective at producing a depletion in the alveolar macrophage population at relatively low IADs and that chronic effects on the cells can be produced by higher concentrations.

The development and interlobar distribution of plutonium-induced pulmonary fibrosis in mice.

Six-week-old mice were exposed by inhalation to an aerosol of 239PuO2 (activity median aerodynamic diameter 2.2 microns) to establish mean alveolar depositions at 2 days after exposure of 4, 40, and 930 Bq of 239Pu. Animals were killed serially after 3, 6, 12, and 18 months at which times the development of the pulmonary fibrotic lesion was assessed by both biochemical and histopathological techniques. Individual measurements of both fresh and dry weights, protein, DNA, and hydroxyproline were made on whole lung and also on each of the five constituent lobes. Early and sustained increases in lung mass, lung protein, and total lung collagen were found, together with a depression of the total cellularity of the lung at 6 and 12 months after exposure. Although at later times compensatory hypertrophy of less affected areas distorted the relationship, systematic trends in the severity of responses between lobes were found. These trends were related to the initial lobar concentrations of 239Pu.

Retention of 239Pu in the mouse lung and estimation of consequent dose following inhalation of sized 239PuO2.

The retention of 239Pu in the lungs of SAS/4 mice following inhalation exposure to sized 239PuO2 particles is described. When the initial alveolar deposition (IAD) was less than 200 Bq, retention of 239Pu could be described by a two-component exponential expression, about 90% being cleared with a half-time of about 40 days and the remainder with a half-time of about 150 days. Similar amounts of 239Pu were retained up to 3 months with IADs greater than 800 Bq, but clearance was impaired thereafter, the half-time of the second component increasing to about 720 days. The retention of 239Pu was independent of the particle size of the administered 239PuO2. Studies of the retention of 239Pu by individual lobes indicated that there were intrinsic interlobar differences which were enhanced at higher IADs. Lung clearance was also studied by the measurement of 239Pu in feces excreted by groups of mice in the period immediately prior to sacrifice. The estimation of radiation dose to lung is discussed.

Dose-dependent effects in the subacute response of the rat lung to quartz. I. The cellular response and the activity of lactate dehydrogenase in the airways.

The response of the rat lung to a range of doses of quartz at 50 and 100 days after its administration by intratracheal instillation was investigated by bronchopulmonary lavage. The effects on the numbers of polymorphonuclear leucocytes, lymphocytes and macrophages recovered are described. In addition, the size distribution of the recovered macrophages was followed and measurements of total numbers of macrophages in the lung were made using a radioactive tracer technique. Finally, the activity of the cytoplasmic enzyme, lactate dehydrogenase, in lavage fluid supernatants was recorded. There was a dose-dependent increase in the recovery of all three cell types and an increase in the total numbers of macrophages in the lungs which showed no signs of resolution over the 100 day period studied. Increasing levels of lactate dehydrogenase were also dose-dependent and well correlated with the numbers of polymorphs and lymphocytes recovered. The importance of this prolonged inflammatory reaction in relation to the fibrogenic action of quartz is discussed.

Dose-dependent effects in the subacute response of the rat lung to quartz. II. Protease activities and levels of soluble hydroxyproline in lung lavage.

The response of the rat lung to a range of doses of quartz at 50 and 100 days after its administration by intratracheal instillation has been assessed by bronchopulmonary lavage. The effects on the levels of protein and hydroxyproline and on the PZ peptidase and collagenolytic activities of lavage fluid supernatants are described and an assessment of the hydroxyproline content of recovered cells is made. In addition, assays for PZ peptidase and collagenase activity were carried out on polymorph enriched fractions and on samples obtained from short-term culture of recovered macrophages. Both protein and hydroxyproline measurements did show dependence on the amount of quartz instilled and the time after its administration. Measurements of PZ peptidase and collagenase activities suggest that the polymorphonuclear leukocytes, not the macrophages, are the major source of these degradative enzymes. The relevance of these findings with regard to the importance of the PMN in quartz-induced fibrosis, is discussed.

Evidence for a dose-dependent inflammatory response to quartz in the rat lung and its significance in early changes in collagen metabolism.

The response of the rat lung to a range of doses of quartz at 50 and 100 days after its administration by intratracheal instillation has been assessed by bronchopulmonary lavage. The effects on the number of polymorphonuclear leukocytes (PMN), lymphocytes and macrophages are described. In addition the concentrations of soluble protein and hydroxyproline and the activities of lactate dehydrogenase, PZ peptidase and collagenase in lavage fluid supernatants were measured and an assessment of the hydroxyproline content of recovered cells was made. Finally PZ peptidase and collagenase were assayed in PMN-enriched cell fractions and in samples obtained from short-term culture of recovered macrophages. There was a dose-dependent increase in the recovery of all three cell types, and in the amounts of lactate dehydrogenase, protein and hydroxyproline in lavage fluids, which showed no signs of resolution over the 100-day period studied. Measurements of PZ peptidase and collagenase suggested that the PMN, not the macrophages, are the major source of these degradative enzymes. The relevance of these findings with regard to the importance of PMN in quartz-induced fibrosis is discussed.

The advantages and limitations of an in vivo test system for investigating the cytotoxicity and fibrogenicity of fibrous dusts.

The acute response of the rat lung to a range of fibrous materials has been investigated by bronchopulmonary lavage, at dose levels of 0.5 and 1.0 mg, 1 and 7 days after their administration by intratracheal instillation. The materials chosen for study included UICC chrysotile A, amosite, crocidolite and anthophyllite, and samples of S. African "long" amosite and glass fiber. In addition, the subacute response to 1, 2 and 3 mg of chrysotile and amosite has been studied at 50 and 100 days after instillation. In the acute phase at 1 day after instillation, the response to chrysotile was greater than that to any of the other materials, but by 7 days there was no gradation in the response to different dusts. In the subacute phase, cell recoveries were low, and it was not possible to assess the long-term cytotoxic or fibrogenic effects of amosite and chrysotile by analyses of lung washes, even though biochemical and histological methods indicated gross changes in lung pathology.

Alveolar deposition of sized particles of 239PuO2 in the mouse.

Data on the alveolar deposition of 239PuO2 particles in the mouse are presented. It is shown that alveolar deposition falls with increasing activity median aerodynamic diameter (AMAD) over the range 0.6 to 2.4 microns. In the mouse, any peak in alveolar deposition must occur at an AMAD of less than 1 micron. Information is also provided on the relative lobar concentrations (RLCs) of 239PuO2. The RLC is greatest in the right apical lobe and increases quite sharply with AMAD. The RLCs of the other lobes of the right lung decline with AMAD. The RLC in the left lobe is relatively unaffected by AMAD.