Oxidative stress levels and dynamic changes in mitochondrial gene expression in a radiation-induced lung injury model

Abstract

The purpose of this study was to set up a beagle dog model, for radiation-induced lung injury, that would be able to supply fresh lung tissues in the different injury phases for research into oxidative stress levels and mitochondrial gene expression. Blood serum and tissues were collected via CT-guided core needle biopsies from dogs in the various phases of the radiation response over a 40-week period. Levels of reactive oxygen species (ROS) and manganese superoxide dismutase 2 (MnSOD) protein expression in radiation-induced lung injury were determined by in situ immunocytochemistry; malondialdehyde (MDA) content and reductase activity in the peripheral blood were also tested; in addition, the copy number of the mitochondrial DNA and the level of function of the respiratory chain in the lung tissues were assessed. ROS showed dynamic changes and peaked at 4 weeks; MnSOD was mainly expressed in the Type II alveolar epithelium at 8 weeks; the MDA content and reductase activity in the peripheral blood presented no changes; the copy numbers of most mitochondrial genes peaked at 8 weeks, similarly to the level of function of the corresponding respiratory chain complexes; the level of function of the respiratory chain complex III did not peak until 24 weeks, similarly to the level of function of the corresponding gene Cytb. Radiation-induced lung injury was found to be a dynamically changing process, mainly related to interactions between local ROS, and it was not associated with the levels of oxidative stress in the peripheral blood. Mitochondrial genes and their corresponding respiratory chain complexes were found to be involved in the overall process.

Keywords: ROS; biopsy; mitochondrial respiratory complex; mtDNA; radiation-induced lung injury.

Fig. 1.

Dynamic changes in the amount of superoxide anion: (A) only very small amounts of superoxide anion showed in the control group; (B) 4 weeks after radiation; (C) 8 weeks after radiation; and (D) 24 weeks after radiation. (E) Compared with the control group, the irradiated samples showed dynamic changes, peaking at 4 weeks, then gradually decreasing over time. At 4 weeks and 8 weeks, the P values were both <0.05.

Fig. 2.

Dynamic changes in the amount of hydrogen peroxide and hydroxyl free radical: (A) only very small amounts of hydrogen peroxide and hydroxyl free radical showed in the control group; (B) 4 weeks after radiation; (C) 8 weeks after radiation; and (D) 24 weeks after radiation. (E) Compared with the control group, the irradiated samples showed dynamic changes, peaking at 4 weeks, then gradually decreasing over time. At 4 weeks and 8 weeks, the P values were both <0.05.

Fig. 3.

Dynamic changes in MnSOD protein expression in lung tissues of beagles with radiation-induced lung injury: (A) the lung tissues of the non-irradiated group showed almost no expression of MnSOD; (B) 4 weeks after radiation, MnSOD was mainly expressed in the bronchial epithelium; (C) 8 weeks after radiation, MnSOD was still mainly expressed in the bronchial epithelium; (D) 8 weeks after radiation, MnSOD was mainly expressed in the Type II alveolar epithelium; (E) 24 weeks after radiation, MnSOD expression was decreased and was primarily expressed in Type II alveolar epithelial cells; (F) negative control group.

Fig. 4.

(A) Dynamic changes in the MDA content in the peripheral blood of beagles with radiation-induced lung injury. (B–E) Dynamic changes in the reductase activity in the peripheral blood of beagles with radiation-induced lung injury: (B) dynamic changes in T-SOD enzyme activity; (C) dynamic changes in CuZnSOD enzyme activity; (D) dynamic changes in MnSOD enzyme activity; (E) dynamic changes in GSHPX enzyme activity.

Fig. 5.

Changes in the copy number of mtDNA in the lung tissues of beagles with radiation-induced lung injury. The copy numbers of the 12S rRNA, ND4, Cox2 and ATP6 genes peaked at 8 weeks after irradiation and then decreased at 24 weeks, whereas the copy numbers of the Cytb gene increased continuously and peaked at 24 weeks after irradiation. The copy numbers of mitochondrial genes in the lungs of the control group did not differ from one time point to another. Three independent biological replicates were used. *P < 0.05.

Fig. 6.

The activity levels of Complex I, III, IV and V exposed to irradiation compared with the activity levels in sham-irradiated mitochondria. The changes in the activity levels of respiratory chain complexes I, IV and V first declined (after 4 weeks), and then peaked at 8 weeks. The activity level of respiratory chain complex III did not peak until 24 weeks after irradiation. Three independent biological replicates were used. *P < 0.05.