Protein Oxidation in the Lungs of C57BL/6J Mice Following X-Irradiation

Abstract

Damage to normal lung tissue is a limiting factor when ionizing radiation is used in clinical applications. In addition, radiation pneumonitis and fibrosis are a major cause of mortality following accidental radiation exposure in humans. Although clinical symptoms may not develop for months after radiation exposure, immediate events induced by radiation are believed to generate molecular and cellular cascades that proceed during a clinical latent period. Oxidative damage to DNA is considered a primary cause of radiation injury to cells. DNA can be repaired by highly efficient mechanisms while repair of oxidized proteins is limited. Oxidized proteins are often destined for degradation. We examined protein oxidation following 17 Gy (0.6 Gy/min) thoracic X-irradiation in C57BL/6J mice. Seventeen Gy thoracic irradiation resulted in 100% mortality of mice within 127-189 days postirradiation. Necropsy findings indicated that pneumonitis and pulmonary fibrosis were the leading cause of mortality. We investigated the oxidation of lung proteins at 24 h postirradiation following 17 Gy thoracic irradiation using 2-D gel electrophoresis and OxyBlot for the detection of protein carbonylation. Seven carbonylated proteins were identified using mass spectrometry: serum albumin, selenium binding protein-1, alpha antitrypsin, cytoplasmic actin-1, carbonic anhydrase-2, peroxiredoxin-6, and apolipoprotein A1. The carbonylation status of carbonic anhydrase-2, selenium binding protein, and peroxiredoxin-6 was higher in control lung tissue. Apolipoprotein A1 and serum albumin carbonylation were increased following X-irradiation, as confirmed by OxyBlot immunoprecipitation and Western blotting. Our findings indicate that the profile of specific protein oxidation in the lung is altered following radiation exposure.

Keywords: 2-D gel electrophoresis; OxyBlot; mass spectrometry; protein carbonylation; pulmonary fibrosis; radiation pneumonitis; thoracic irradiation.

Figure 1

Seventeen Gy thoracic irradiation is sufficient to induce pulmonary fibrotic remodeling within 200 days (A) Kaplan-Meier plot of survival following 14, 16, 17, or 18 Gy of thoracic irradiation (N = 7–8); (B) In a separate experiment, lung tissue was obtained from sham-irradiated and irradiated mice exposed to 17 Gy thoracic irradiation, at 180 days postirradiation. Tissues were fixed, paraffin embedded, and used for Masson’s Trichrome stain. Collagen appears as blue. Representative data are shown (N = 6).

Figure 2

Two dimensional gel electrophoresis and OxyBlot analysis of sham-irradiated and irradiated lung proteins. C57BL/6J mice were either sham irradiated (control, A,B) or exposed to 17 Gy thoracic irradiation (17 Gy, C,D). Lung cell lysates were obtained 24 h postirradiation and protein lysates from individual mice for used for one gel. Upper panels: OxyBlots (A,C). Lower panels: Coomassie stained gels (B,D). Proteins detected by OxyBlot are indicated with numbered arrows. OxyBlots and gels are representative of N = 4 gels per group.

Figure 3

Confirmation of carbonylation on serum albumin and apolipoprotein A1. C57BL/6J mice were sham irradiated (control) or exposed to 17 Gy thoracic irradiation. Lung cell lysates were obtained 24 h postirradiation from three animals per group and DNP-labeled for immunoprecipitation with the OxyBlot antibody. Immunoprecipitated proteins were then Western blotted for (A) serum albumin or (B) apolipoprotein A1. Bar indicates average of band densitometry of three animals per condition; * indicates p < 0.05.