Flaxseed Mitigates Acute Oxidative Lung Damage in a Mouse Model of Repeated Radiation and Hyperoxia Exposure Associated with Space Exploration

Abstract

Background: Spaceflight missions may require crewmembers to conduct extravehicular activities (EVA). Pre-breathe protocols in preparation for an EVA entail 100% hyperoxia exposure that may last for a few hours and be repeated 2-3 times weekly. Each EVA is associated with additional challenges such as low levels of total body cosmic/galactic radiation exposure that may present a threat to crewmember health. We have developed a mouse model of total body radiation and hyperoxia exposure and identified acute damage of lung tissues. In the current study we evaluated the usefulness of dietary flaxseed (FS) as a countermeasure agent for such double-hit exposures.

Methods: We evaluated lung tissue changes 2 weeks post-initiation of exposure challenges. Mouse cohorts (n=5/group) were pre-fed diets containing either 0% FS or 10% FS for 3 weeks and exposed to: a) normoxia (Untreated); b) >95% O₂ (O₂); c) 0.25Gy single fraction gamma radiation (IR); or d) a combination of O₂ and IR (O₂+IR) 3 times per week for 2 consecutive weeks, where 8-hour hyperoxia treatments were spanned by normoxic intervals.

Results: At 2 weeks post challenge, while control-diet fed mice developed significant lung injury and inflammation across all challenges, FS protected lung tissues by decreasing bronchoalveolar lavage fluid (BALF) neutrophils (p<0.003) and protein levels, oxidative tissue damage, as determined by levels of malondialdehyde (MDA) (p<0.008) and nitrosative stress as determined by nitrite levels. Lung hydroxyproline levels, a measure of lung fibrosis, were significantly elevated in mice fed 0% FS (p<0.01) and exposed to hyperoxia/radiation or the combination treatment, but not in FS-fed mice. FS also decreased levels of a pro-inflammatory, pro-fibrogenic cytokine (TGF-β1) gene expression levels in lung.

Conclusion: Flaxseed mitigated adverse effects in lung of repeat exposures to radiation/hyperoxia. This data will provide useful information in the design of countermeasures to early tissue oxidative damage associated with space exploration.

Keywords: Acute lung injury; Bronchoalveolar lavage; Double-hit; Hyperoxia; Inflammation; Lung fibrosis; Mouse model; Nitrosative stress; Oxidative stress; Radiation pneumonopathy; Space exploration; TGF-β1; Total body irradiation; extravehicular activity.

Figure 1

Experimental Plan of In Vivo Animal Exposure. Mouse cohorts (n=5/group) were fed 0% or 10% FS diet for 3 weeks prior to exposure and remained on test diets throughout the course of the study. Mice were exposed to a double-hit challenge of 0.25 Gy total body ionizing gamma irradiation (IR) and 100% pO₂ for 8 hours followed by intermittent normoxia (21% pO₂). This cycle was performed 3 times a week for 2 weeks. Mice were sacrificed after 2 weeks of challenge exposure. A total of 24 hours of hyperoxia/radiation exposure weekly simulates the maximum allowed weekly extravehicular activity (EVA) exposure for crewmembers on lengthy spaceflights.

Figure 2

Detection of Mammalian Lignans in Mouse Plasma. Separate cohorts of mice (n=5/group) were fed 0% or 10% FS diet and exposed to 100% O₂ for 8 hours only (O₂), 0.25 Gy total body ionizing gamma irradiation (IR) only, or a double-hit combination of both challenges (O₂+IR) followed by intermittent normoxia (21% pO₂) for repeated cycles 3 times a week for 2 weeks. Mice were sacrificed after 2 weeks of challenge exposure and plasma analyzed for levels of circulating mammalian lignans enterodiol (Panel A) and enterolactone (Panel B) using GC/MS/MS. Data is represented as mean ± SEM.

Figure 3

Flaxseed ameliorates increased Hyperoxia, Radiation, or Double-Hit Combination Challenge Induced Lung Inflammation and Proinflammatory Cytokine Release. Separate cohorts of mice (n=5/group) were fed 0% or 10% FS diet and exposed to 100% O₂ for 8 hours only (O₂), 0.25 Gy total body ionizing gamma irradiation (IR) only, or a double-hit combination of both challenges (O₂+IR) followed by intermittent normoxia (21% pO₂) for repeated cycles 3 times a week for 2 weeks. Mice were sacrificed after 2 weeks of challenge exposure. BALF was evaluated for total neutrophil cell counts (Panel A) and levels of pro-inflammatory cytokines, IL-1β (Panel B) and TNFα (Panel C). Data is represented as mean ± SEM. *p< 0.05 for O₂, IR, and O₂+IR versus respective untreated control mice in each diet cohort. #p< 0.05 for respective 0% FS fed mice exposed to O₂, IR, and O₂+IR versus 10% FS fed mice exposed to O₂, IR, and O₂+IR.

Figure 4

Flaxseed mitigates increased Oxidative Stress in Lung Tissue and BALF due to Hyperoxia, Radiation, or Double-Hit Combination Challenge Separate cohorts of mice (n=5/group) were fed 0% or 10% FS diet and exposed to 100% O₂ for 8 hours only (O₂), 0.25 Gy total body ionizing gamma irradiation (IR) only, or a double-hit combination of both challenges (O₂+IR) followed by intermittent normoxia (21% pO₂) for repeated cycles 3 times a week for 2 weeks. Mice were sacrificed after 2 weeks of challenge exposure. Levels of malondialdehyde, a marker of lipid peroxidation, were determined in mouse lung tissue (Panel A) and BALF (Panel B). Data is represented as mean ± SEM. *p< 0.05 for O₂, IR, and O₂+IR versus respective untreated control mice in each diet cohort. #p< 0.05 for 0% FS fed mice exposed to O₂, IR, and O₂+IR versus respective 10% FS fed mice exposed to O₂, IR, and O₂+IR.

Figure 5

Flaxseed Abrogates increased Nitrosative Stress from Exposure to Hyperoxia, Radiation, or Double-Hit Combination Challenge Separate cohorts of mice (n=5/group) were fed 0% or 10% FS diet and exposed to 100% O₂ for 8 hours only (O₂), 0.25 Gy total body ionizing gamma irradiation (IR) only, or a double-hit combination of both challenges (O₂+IR) followed by intermittent normoxia (21% pO₂) for repeated cycles 3 times a week for 2 weeks. Mice were sacrificed after 2 weeks of challenge exposure. Levels of nitrite, a stable breakdown product of nitric oxide, were determined in mouse BALF (Panel A) and plasma (Panel B). Data is represented as mean ± SEM. *p< 0.05 for O₂, IR, and O₂+IR versus respective untreated control mice in each diet cohort. #p< 0.05 for 0% FS fed mice exposed to O₂, IR, and O₂+IR versus respective 10% FS fed mice exposed to O₂, IR, and O₂+IR.

Figure 6

Flaxseed blunts increased Lung Fibrosis and Profibrogenic Cytokine Levels induced by Exposure to Hyperoxia, Radiation, or Double-Hit Combination Challenge Separate cohorts of mice (n=5/group) were fed 0% or 10% FS diet and exposed to 100% O₂ for 8 hours only (O₂), 0.25 Gy total body ionizing gamma irradiation (IR) only, or a double-hit combination of both challenges (O₂+IR) followed by intermittent normoxia (21% pO₂) for repeated cycles 3 times a week for 2 weeks. Mice were sacrificed after 2 weeks of challenge exposure. Lung tissue was harvested and evaluated for hydroxyproline content, a measure of fibrosis (Panel A). Changes in lung tissue mRNA levels of profibrogenic TGFβ1 were determined quantitative realtime PCR analysis (Panel B). Analysis was performed in duplicate and gene expression normalized to 18S ribosomal RNA. Levels of active TGFβ1 were determined in mouse BALF using ELISA (Panel C). All data is represented as mean ± SEM. *p< 0.05 for O₂, IR, and O₂+IR versus respective untreated control mice in each diet cohort. #p< 0.05 for 0% FS fed mice exposed to O₂, IR, and O₂+IR versus respective 10% FS fed mice exposed to O₂, IR, and O₂+IR.

Figure 7

Flaxseed blunts increased Lung Fibrosis induced by Exposure to Hyperoxia, Radiation, or Double-Hit Combination Challenge-A histological evaluation Representative lung photomicrographs are shown here, from the mouse cohorts described in Figure 6. Sections are stained with Mason's Trichrome Blue to identify collagen deposition. Magnification, 400×.