Spaceflight missions may require crewmembers to conduct extravehicular activities (EVA) for repair, maintenance or scientific purposes. EVAs should total no more than 24 hours weekly per crew member. During each EVA, crewmembers are subjected to a number of challenges that may induce oxidative tissue damage and thus present a threat to their health and therefore pose a limitation to the success of the mission. One such challenge is the exposure to 100% oxygen as well as to low levels of total body cosmic/galactic radiation that crewmembers will be exposed to during an EVA. Currently, no studies have addressed this “double hit” hyperoxia/radiation effect so we developed and characterized a novel, mouse model of repeated total body radiation and hyperoxia exposure. Preliminary studies identified acute lung damage associated with such treatment so the goal of the proposed study is to further characterize the acute damage and most importantly, to provide information on the long-term effects of such an exposure (Aim 1). Additionally, initial findings suggested that dietary administration of whole grain dietary flaxseed (FS) ameliorated adverse acute effects in the same model. Our group was the first to show in murine models of radiation and hyperoxic lung injury the protective properties of FS. Importantly, lignan phenolics in FS such as secoisolariciresinol diglucoside (SDG), are known antioxidant, anti-inflammatory and anticarcinogenic agents. We hypothesize that FS and FS lignans will be effective countermeasures of this double-hit toxicity to lung tissues (Aim 2). We further explored the usefulness of FS and SDG in both acute and chronic oxidative lung damage in this model. FS and specifically SDG are safe, readily available and thus, attractive candidates to evaluate as oxidative damage countermeasures in the proposed double hit radiation/hyperoxia model. Data from this work are relevant to EVA-related pathophysiology associated with space exploration.
AIM 1: Mouse cohorts (n=5-15/group) were exposed to repeated: a) normoxia; b) >95% O2 (O2); c) 0.25Gy single fraction gamma radiation (IR); or d) a combination of O2 and IR (O2+IR) given 3 times per week for 4 weeks. Lungs were evaluated for oxidative damage, active TGFI1 levels, cell apoptosis, inflammation, injury, and fibrosis at 1, 2, 4, 8, 12, 16, and 20 weeks post-initiation of exposure.
AIM 2: To test the usefulness of flaxseed in ameliorating acute side effects of radiation/hyperoxia in lung following a short term exposure (2 weeks), mouse cohorts (n=5/group) were pre-fed diets containing either 0% FS or 10% FS for 3 weeks and exposed to: a) normoxia; b) >95% O2 (O2); c) 0.25Gy single fraction gamma radiation (IR); or d) a combination of O2 and IR (O2+IR). The stress challenge (O2, IR or a combination of both) was given 3 times per week for 2 consecutive weeks and consisted of 8-hour hyperoxia treatments spanned by normoxic intervals each time. Cohorts that received TBI were exposed to gamma radiation shortly prior to entering the hyperoxic chambers. Lungs were evaluated for oxidative damage, inflammation, injury and fibrosis after 2 weeks of exposure to determine acute lung changes.
To test the usefulness of flaxseed and its lignan component (FLC) enriched in SDG in ameliorating late effects (16 weeks) of radiation/hyperoxia in lung following prolonged exposure (4 weeks), mouse cohorts were fed 0% FS, 10% FS, and 10% FLC. Along with evaluating chronic lung changes, lung inflammation, and lung oxidative stress, we also evaluated changes in mouse bodyweight and blood PaO2 16 weeks after the initiation of exposure conditions.
Two separate series of experiments were performed based on the Specific Aims that describe our experimental approach:
In the first series, we determined acute and chronic lung damage in our mouse model of hyperoxia/radiation exposure relevant to EVA. The results are summarized below:
After 4 weeks of treatment, mice exposed to all challenges (O2, IR, O2+IR) displayed significantly (p<0.05) reduced bodyweight compared to normoxic, untreated control mice for each respective time point (1,2 months-3,4 still pending). This reduction in bodyweight was obvious as early as 1 month and persisted throughout the duration of the experiment. Physiological parameters related to pulmonary function, such as blood oxygenation, were significantly (p<0.01) affected by all stress challenges. At 1 and 2 months post-challenge, mice exposed to a single challenge (O2 or IR) had reduced blood oxygenation levels (94% and 92% PaO2 in O2 and IR mice, respectively vs. 97% PaO2 in control mice). Importantly, mice exposed to the combination treatment (O2+IR) displayed more robust (p<0.001) deterioration of oxygen saturation (88.3% and 88.7% PaO2) at 1 and 2 months post-exposure, respectively. All treatment challenges (O2, IR, O2+IR) resulted in significant oxidative lung damage (p<0.05) evidenced by increased concentrations of malondialdehyde (MDA) at both 1 and 2 months post-exposure. Remarkably, lung MDA content as compared to untreated controls was increased significantly by 282%, 121%, and 123% for O2, IR, and O2+IR, respectively. Significant elevation of lung tissue fibrosis (p<0.05), determined by lung hydroxyproline content, was detected as early as 1 month post-exposure in mice exposed to all challenge conditions (O2, IR, O2+IR). Specifically, lung tissue hydroxyproline content increased by 43%, 27%, and 25% for O2, IR, and O2+IR,respectively as compared to untreated controls.
Conclusion: Characterized significant, chronic lung changes in our murine model of repeated radiation and hyperoxia exposure relevant to space travel. Lung tissue changes, detectable several months after the original exposure, include significant oxidative lung damage (lipid peroxidation) and increased pulmonary fibrosis. These findings, along with the observed decreases in blood oxygenation levels in all challenge conditions (whether single or in combination), lead us to conclude that in our model of repeated exposure to oxidative stressors, chronic tissue changes are detected that persist even months after the exposure to the stressor has ended. This data will provide useful information in the design of countermeasures to tissue oxidative damage associated with space exploration. Data are published in Pulmonary and Respiratory Medicine,3(5), P 1000158, 2013. PMID:24358450
In a second series of experiments, we determined whether dietary adminsitreation of flaxseed, would ameliorate the adverse effects of hyperoxia/radiation exposure in this mouse model. The results are summarized below.
0% FS-fed mice developed significant lung injury and inflammation across all challenges, as evidenced by bronchoalveolar lavage neutrophils (p<0.003) and increased protein levels as well as increased oxidative tissue damage (p<0.008), as determined by lung malondialdehyde content. Lung hydroxyproline content increased in 0% FS-fed mice exposed to IR and O2+IR (p<0.001). All adverse effects due to challenge conditions were ameliorated in FS-fed mice. Lung hydroxyproline levels were significantly elevated in mice fed 0% FS and exposed to hyperoxia/radiation or the combination treatment, but not in flaxseed-fed mice. To determine whether FS altered levels of a pro-inflammatory cytokine (TGFB1) we looked at gene expression levels in lung. We determined a significant 1.61, 1.55, and 2.55 fold change induction of TGFB1 over control in mice fed 0% FS and exposed to O2, IR, and O2+IR, respectively. Flaxseed abrogated the challenge induced increase in TGFB1 in all challenge conditions. We determined blood PaO2 and hydroxyproline levels, as a measure of lung fibrosis, at the time of sacrifice of each cohort, at 4 months post-challenge and observed no significant change from untreated control mice. Notably, however, mice fed 10% FS or 10% FLC improved lung inflammation, as determined by BALF WBCs. Cytokine levels in BALF were low (below the lower limit of detection of the assay), however, IL-10, a cytokine with known anti-inflammatory properties, was significantly elevated in BALF from FS and FLC fed mice exposed to combination treatment (O2+IR).