Intratracheal injection of adenovirus containing the human MNSOD transgeneprotects athymic nude mice from irradiation-induced organizing alveolitis

Purpose: A dose and volume limiting factor in radiation treatment of thorac ic cancer is the development of fibrosis in normal lung. The goal of the pr esent study was to determine whether expression prior to irradiation of a t ransgene for human manganese superoxide dismutase (MnSOD) or human copper/z inc superoxide dismutase (Cu/ZnSOD) protects against irradiation-induced lu ng damage in mice. Methods and Materials: Athymic Nude (Nu/J) mice were intratracheally inject ed with 10(9) plaque-forming units (PFU) of a replication-incompetent mutan t adenovirus construct containing the gene for either human MnSOD, human co pper/zinc superoxide dismutase (Cu/ZnSOD) or LacZ. Four days later the mice were irradiated to the pulmonary cavity to doses of 850, 900, or 950 cGy. To demonstrate adenoviral infection, nested reverse transcriptase-polymeras e chain reaction (RT-PCR) was carried out with primers specific for either human MnSOD or Cu/ZnSOD transgene on freshly explanted lung, trachea, or al veolar type II cells, and immunohistochemistry was used to measure LacZ exp ression. RNA was extracted on day 0, 1, 4, or 7 after 850 cGy of irradiatio n from lungs of mice that had previously received adenovirus or had no trea tment. Slot blot analysis was performed to quantitate RNA expression for IL -1, tumor necrosis factor (TNF)-alpha, TGF-beta, MnSOD, or Cu/ZnSOD. Lung t issue was explanted and tested for biochemical activity of MnSOD or Cu/ZnSO D after adenovirus injection. Other mice were sacrificed 132 days after irr adiation, lungs excised, frozen in OCT, (polyvinyl alcohol, polyethylene gl ycol mixture) sectioned, H&E stained, and evaluated for percent of the lung demonstrating organizing alveolitis. Results: Mice injected intratracheall y with adenovirus containing the gene for human MnSOD had significantly red uced chronic lung irradiation damage following 950 cGy, compared to control mice or mice injected with adenovirus containing the gene for human Cu/ZnS OD or LacZ. Immunohistochemistry for LacZ protein in adenovirus LacZ (Ad-La cZ)-injected mice demonstrated expression of LacZ in both the upper and low er airway. Nested RT-PCR showed lung expression of MnSOD and Cu/ZnSOD for a t least 11 days following infection with each respective adenovirus constru ct. Nested RT-PCR using printers specific for human MnSOD demonstrated incr eased expression of the human MnSOD transgene in the trachea and alveolar t ype II cells 4 days after virus injection on the day of irradiation. At thi s time point, increased biochemical activity of MnSOD and Cu/ZnSOD respecti vely, was detected in lungs from these two adenovirus groups, compared to e ach other or to control or adenovirus LacZ mice. Slot blot analysis of RNA from lungs of mice in each group following 850 cGy irradiation demonstrated decreased expression of mRNA for interleukin-I (IL-1), TNF-alpha, and tran sforming growth factor-beta (TGF-beta) in the MnSOD adenovirus-injected mic e, compared to irradiated control, LacZ, or Cu/ZnSOD adenovirus-injected, i rradiated mice. Mice receiving adenovirus MnSOD showed decreased organizing alveolitis at 132 days in all three dose groups, compared to irradiated co ntrol or Ad-LacZ, or Ad-Cu/ZnSOD mice. Conclusions: Overexpression of MnSOD in the lungs of mice prior to irradiat ion prevents irradiation-induced acute and chronic damage quantitated as de creased levels of mRNA for IL-1, TNF-alpha, and TGF-beta in the days immedi ately following irradiation, and decrease in the percent of lung demonstrat ing fibrosis or organizing alveolitis at 132 days. These data provide a rat ional basis for development of gene therapy as a method of protection of th e normal lung from acute and chronic sequellae of ionizing irradiation. (C) 1998 Elsevier Science Inc.