To correlate misonidazole concentrations and oxygen pressures (Po2) at identical locations within EMT6/Ro multi-cell spheroids (mean diameters +/- SD: 867 +/- 20 microns), Po2 measurements were performed with oxygen-sensitive microelectrodes during incubation of these spheroids with tritiated misonidazole (10 mg/I; 445 microCi/mg). In each individual spheroid, Po2 profiles were correlated with the corresponding spatial distribution of misonidazole as quantified by conventional autoradiography and grain counting. To compare the oxygenation status of spheroids in the measuring chamber with that of spheroids in spinner culture, misonidazole labeling was performed in both environments following the same protocol. All experiments were conducted in 20% oxygen and BME or in 5% oxygen and DMEM to obtain spheroids with different degrees of oxygenation. Labeled misonidazole was fairly evenly distributed in the outer, better oxygenated regions of EMT6 spheroids. In contrast, there was an accumulation of the labeled substance near central necrosis where low oxygen tensions were measured. Grain densities were similar at corresponding oxygen pressures under both environmental conditions. Except for some scatter, grain density as a function of oxygen pressure showed little variation in the Po2 range of 20-60 mm Hg, but exhibited a steep increase below 10 mm Hg. The findings imply that a substantial rise in local misonidazole labeling indicates a metabolically active tissue region at low Po2 that is not necessarily identical with the radiobiologically hypoxic cell fraction. A comparison of the labeling densities of spheroids in spinner flasks and in the Po2 measuring chamber indicates that oxygenation of spheroids is better in rotation culture than during microelectrode measurements.
CGRAY et al. (1953) suggested that the oxygen concentration in tumour cells might influence the curability of some human cancers by ionizing radiation. Recent clinical investigations using hypoxic cell radiosensitizers (Urtasun et al., 1976), hyperbaric oxygen (Henk & Smith, 1977) and pretherapy transfusion of anaemic patients (Bush et al., 1978) confirnm the postulate that lhypoxia does influence the radiocurability of some tumours. Although the oxygenation status of tumours is considered to be important information for clinical oncologists, no technique is currently available to provide such information for individual tumours.
Strain differences in the intermediate and late phases of the radiation response of mouse lung were investigated histologically. The proportion of lung impairment in mice at 28 and 52 weeks postirradiation and in mice dying of respiratory insufficiency was assessed by scoring lung acini as nonfunctional due to lesions which obstructed airflow, or open and presumably functional. The nine strains tested were divided into three groups on the basis of the late fibrotic response. Group 1 mice, three C57 strains, developed extensive contracted fibrosis and usually showed enough damage to explain late deaths. Group 2, SWR, A, and BALB/c strains, developed foci of contracted fibrosis. Group 3, CBA and two C3H strains, did not form fibrotic scars. Mice in Groups 2 and 3 that died with no pleural effusions appeared to have insufficient late lung damage to account for respiratory distress. Problems with pulmonary blood flow were indicated by evidence of loss of fine vasculature and right ventricular hypertrophy. In nondistressed, late-stage mice in Groups 2 and 3, loss of capillary perfusion in lung parenchyma free of obvious lesions was demonstrated by infusion of colloidal carbon. In one strain, A, an estimate of the proportion of nonperfused lung was made on distressed late-stage mice. Almost 50% of lung acini were nonfunctional as a result of nonperfusion, and an additional 9% of acini were nonfunctional due to lesions obstructing ventilation. It is suggested that nonperfusion of apparently normal lung acini is a major factor in late-phase deaths in those mouse strains which show little or no fibrosis.
Strain differences in the radiation response of mouse lung during the early phase (before 28 weeks postirradiation) were investigated histologically. The nine strains tested were divided into three groups on the basis of the nature of the edema present, the occurrence of hyaline membranes, and the presence of fibrosis. Group 1 mice, three C57 strains, developed hyaline membranes, focal fibrosis, and a protein-rich edema containing fibrin. Group 3, CBA and two C3H strains, had only a protein-poor edema with little fibrin and developed no visible fibrosis. Group 2 mice had both types of edema and small quantities of focal fibrosis. The degree of lung impairment in mice dying of respiratory insufficiency was assessed by scoring lung acini as nonfunctional or open and presumably functional. Over 70% of acini were nonfunctional as a result of airflow obstruction. This was considered sufficient to account for death. Carbon perfusion immediately before sacrifice indicated that all types of lesions were at least partially perfused with blood. Pleural effusions were found in some individuals of two strains. The proportion of nonfunctional acini was similar in mice of the same strain with and without effusions, which would not be expected if the effusions contributed appreciably to respiratory distress in the early phase.
Summary Polyclonal antibodies have been raised in rabbits to a haemocyanin adduct of a reductivelyactivated, fluorinated analogue of misonidazole. Fluorescence immunohistochemical studies show that the polyclonal antibodies bind to spheroid sections and tumour sections in patterns similar to those revealed by autoradiographic studies with a tritium-labelled derivative of the fluorinated misonidazole analogue.
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