Partial volume rat lung irradiation: An evaluation of early DNA damage

Abstract

1. To investigate early DNA damage induced in rat lung cells following single-dose, partial-volume irradiation (lung base and lung apex). 2. To determine the variation in DNA damage in different lung regions. 3. To investigate the possible mechanisms associated with early DNA damage after lung irradiation. The whole lung or the upper or lower half of the entire lung of Sprague-Dawley rats was exposed to 10 Gy 60Co gamma rays. The animals were sacrificed at various times up to 42 h after irradiation. A trypsin-digested lung cell suspension was prepared and cells that attached to slides in the initial 24-h period were then grown in the presence of culture medium with cytochalasin-B for a further 72 h. Radiation-induced DNA damage was quantified in the cells (primarily fibroblasts) from both irradiated and unirradiated lung regions by using a well-characterized micronucleus assay. When the lungs were removed at 16-18 h after whole-lung irradiation, about 0.85 micronuclei (MN) per binucleate cell (BNC) were observed in the lung cells of the irradiated animals, compared to 0.02 MN/BNC in the lung cells of the controls. When only the lung base was irradiated, the frequency of micronuclei was 0.85 MN/BNC compared to 0.43 MN/BNC observed in cells from the irradiated lung apex. Of particular interest was the finding that the unirradiated lung apex also showed a large frequency of micronuclei (0.43 MN/BNC) after the irradiation of the lung base, whereas the unirradiated lung base showed only a marginal (approximately 2-fold) increase relative to the spontaneous frequency following irradiation of the lung apex. The changes in the frequency of micronuclei varied with the time at which the lungs were removed from the rats for early times, but had stabilized by 18 h after irradiation. Normal (unirradiated) cells grown in filtered or unfiltered conditioned media obtained from irradiated cell cultures showed an insignificant marginal increase in the number of micronuclei relative to the spontaneous frequency. Lung cells obtained from the lung base or the lung apex of healthy controls and irradiated separately in vitro showed no regional differences in the induction of micronuclei. Cells from the lungs of rats injected with superoxide dismutase, within 1 h prior to irradiation of the lung base, and processed 16-18 h after irradiation showed a reduction in the number of MN in the shielded lung apex, indicating the possible involvement of oxygen radicals. These data indicate that cells in the lung base sustain more DNA damage than those in the lung apex when either region is irradiated; however, when the whole lung, is irradiated, the lung damage observed is similar in the two regions. Also, out-of-field effects are observed for the lung apex but not the lung base. Possible mechanisms include a clastogenic (chromosome damaging) factor produced in the plasma following irradiation and/or the production of oxygen radicals by activated lymphocytes/monocytes. The partial blocking of the DNA damage, observed in the unirradiated lung apex following irradiation of the lung base, by superoxide dismutase, suggests that oxygen radicals are involved in this out-of-field effect. These radicals are likely produced as a result of the induction of inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) by the irradiation. The reason for the lack of an out-of-field effect in the lung base when the lung apex is irradiated is unknown, but may be due to the greater volume of lung irradiated in the lower lung field, because this is likely to affect the level of cytokines produced. Alternatively, it may reflect cytokines produced as a result of the partial liver irradiation that occurs with the lower lung field.