We demonstrate here for the first time the role of FS in boosting survival and mitigating the acute and chronic damage induced by X-ray radiation exposure of lung tissues when administered days and even weeks after radiation exposure. Results from our study show that FS significantly ameliorates the XRT-induced damage by improving survival and body weight of mice fed with FS not only when diet was given prior to XRT but also when diet was started 2, 4 and 6 weeks after XRT. We also found that FS diet mitigated the deleterious effects of XRT by: a) improving pulmonary hemodynamics and blood oxygenation levels, b) decreasing lung injury by lowering BAL protein levels, c) reducing pulmonary fibrosis by decreasing collagen content of lung tissues, d) reducing lung inflammation by decreasing WBC influx into the airways and by e) oxidative modification of mouse lungs as evidenced by levels of lipid peroxidation. BAL cytokine analysis, moreover, pointed to an alteration of the chronic inflammatory profile of irradiated lungs favoring a mitigated radiation effect as a result of the FS diet.
Some reports suggest using lung lavage to remove radionuclides inhaled after a dirty bomb detonation as a possible countermeasure [28
]. However, this remains an impractical countermeasure since multiple lavages may be required for efficient removal of radionuclide burden while the procedure itself is associated with known risks of an invasive procedure that requires anesthesia. Alternatively, a plethora of compounds both chemical/natural are being evaluated with the intent of mitigating radiation damage [17
]. To date Amifostine is the only FDA approved cytoprotective radiation mitigator. However, the use of Amifostine has been limited by its significant systemic toxicity [17
]. Further, most of the compounds that offered a positive radioprotection on cells have not shown efficacy in pre-clinical animal studies.
An ideal radiation mitigator should be safe, effective, have a long shelf life and an easy route of administration. Flaxseed, due to its high content of lignans and omega-3 fatty acids, is a dietary supplement that has numerous medicinal, anti-inflammatory and antioxidant properties. FS and its bioactive components have been extensively studied for their anti-inflammatory [11
], anticarcinogenic [30
] and anti-atherogenic effects [32
] in several organ systems. Importantly, prolonged FS administration has not been associated with any significant toxicity [33
]. Therefore, we hypothesized that flaxseed may be an effective, safe and cost-effective mitigator of the radiation damage.
Our data showed that 10%FS diet supplementation significantly increased the survival in mice in all the irradiated groups (Figure ) irrespective of the time of initiation of the FS diet (70-88% survival) as compared to the survival of mice fed with 0%FS diet (40% survival). It is evident from our results that FS diet protects mice from XRT-induced mortality whether given therapeutically or preventively. Improvement of survival using antioxidants such as N-acetyl-Cysteine (NAC) or mitochondrion-targeted small molecule radiation damage mitigators has been shown in mouse models of abdominal irradiation [34
] or total body irradiation [35
] respectively. To our knowledge our study is the first to report that a mitigator of radiation damage improves survival of animals in an experimental model of thoracic radiation damage. Results revealed radiation-induced increment in lipid peroxidation in lungs (Figure ). Lipid peroxidation (LP) results from a cascade of events induced by radiation in biological membranes. FS diet led to a significant drop off in the LP levels in all the FS-fed experimental mouse groups. Some reports show that other plant extracts also decrease radiation-induced LP [36
]. Recent work by Gauter-Fleckenstein et al
] showed mitigation of radiation-induced oxidative lung tissue changes by an superoxide dismutase (SOD) mimetic, although beneficial effects of the mitigator were limited. Oxidative stress in lungs was only then mitigated when the SOD mimetic was given up to 24 hours post radiation and not when given days (3 days) or weeks (8 weeks) post irradiation. In our work the drop in lipid peroxidation of lung tissues was significant even when the FS diet was initiated 4 and 6 weeks post radiation challenge. Further, our lab is currently exploring dietary formulations of flaxseed components, to identify the chief bioactive ingredient(s) that mitigate radiation effects.
Radiation-induced inflammation is an important side effect that contributes to normal tissue injury [7
]. Our results indicated that XRT-induced lung inflammation and impaired blood oxygenation (decreased Pa
O2) were improved with FS diet when initiated prior to or, importantly, days and weeks after XRT. Vujaskovic and coworkers [38
] have shown that severe hypoxia develops months post an initial radiation exposure of lung tissues. Such hypoxia contributes significantly to the development of a cascade of events leading to lung injury. Improved blood oxygenation of all FS-fed mouse groups (Figure ) may lead to decreased levels of tissue hypoxia and may thus, explain mitigation of adverse radiation effects even when diets are initiated post challenge. In fact, recent work by the same group [37
] using an SOD mimetic to mitigate tissue hypoxia, showed that this would be possible even when given weeks post-irradiation, something which further corroborates with our findings with the FS diet. Decrease of radiation-induced lung inflammation by a mitigator has never been shown. This is the first report that antioxidant agents, such as the FS diets, mitigate pulmonary inflammation when given weeks post initial challenge.
A major feature of radiation pneumonitis is a considerable increase in the alveolar protein exudates, an indicator of increased vascular permeability and direct lung injury [38
]. Radiation causes damage to resident lung cells which in turn release inflammatory mediators (cytokines) and recruit inflammatory cells to the site [39
]. BAL protein level is the most direct and reliable measure of lung injury, translating in actual tissue damage while activation markers of lung tissue inflammatory cell content such as the macrophage ED-1 marker, is a measure rather of inflammation and not injury [37
]. Our results show a significant mitigation of lung injury in all the experimental FS diet fed groups, regardless of the time of FS diet initiation. This may be attributed to decreased inflammatory cell influx and membrane oxidation in FS-supplemented mice. This is the first report that an agent is reported to mitigate Phys
siological lung injury from radiation in vivo
Radiation pneumonitis also involves irreversible fibrotic changes in lung tissues occurring in the late phase of the radiation response [40
]. We have shown that wholegrain FS was protective against experimental radiation fibrosis [22
]. Our current study showed for the first time that fibrotic processes can be blunted in pulmonary tissues even when the protective agent is given post-radiation damage, i.e., as a radiation mitigator. Despite notable benefits of a therapeutic usefulness of FS diet (i.e., when initiated at 0, +2, +4 and +6 weeks post XRT), however, the fact remained that FS-mediated decline in both lung OH-Proline levels and FI was more prominent when diet was started preventively, i.e., 3 weeks prior to XRT. This is the first time that any botanical or chemical agent is indicating mitigation of fibrotic changes effect in lungs.
Radiation exposure leads to histologically recognizable chronic injury initiated by a series of molecular responses involving a number of inflammatory cytokines, pro-fibrotic cytokines and chemokines produced by a variety of cell types, including macrophages, epithelial cells and fibroblasts [27
]. While many studies in rodent thoracic irradiation models focus on cytokine release during the initial post-irradiation phase [41
], studies such as those of Rubin et al
] analyzed the cytokine profile in lungs during the late, chronic phase. In the present study, we evaluated the effect of FS diet on inflammatory cytokines in the late phase of thoracic radiation-exposed mice. As expected, the cytokine levels of the lung tissues and fluids measured in our model reflect levels comparable to those in chronic inflammation and not in acute responses. Several cytokines associated with inflammation were significantly lower in irradiated FS-fed mice as compared to irradiated mice on control diet, while no other cytokines were significantly aggravated by the FS diet. Our studies show, for example, a sustained 3-fold increased BAL level of IL-6 in radiation-exposed mice if fed a control diet while dietary supplementation of flaxseed, given even weeks post initial insult, decreased IL-6 levels, reflecting a low inflammatory state of lung tissues.
Radiation-induced pulmonary injury consists of an early latent period followed by chronic inflammation that leads to collagen deposition and ultimately to fibrosis. Studies suggest that chronic inflammation occurs due to the release of cytokines, chemokines, growth factors and it induces the development of fibrosis. Rubin et al
] reported that a perpetual cascade of cytokines activated soon after radiation exposure that leads to late pulmonary fibrosis.
In the present study we also evaluated the effect of FS diet on inflammatory cytokines in radiation-exposed murine models. Macrophage inflammatory protein 1-α (MIP-1α) appears to be an important cytokine mediator of pulmonary inflammation and injury. Growing pre-clinical and clinical data suggest a potential relationship between serum MIP-1α levels and the risk of lung injury following thoracic radiation.
Increased production of IL-6 is known to occur in the chronic inflammation scenario. It stops production of TNF-α, an acute phase inflammatory cytokine, and is found in chronic disease conditions like thyroditis and type I diabetes. Our studies also show a high level of IL-6 in radiation-exposed mice. Dietary supplementation of flaxseed decreased IL-6 levels, indicating a low inflammatory state. Both IL-4 and IL-17 are known to induce IL-6 production. However in our study, the increase in IL-6 in radiation exposed mice cannot be attributed to IL-17 and IL-4 as both these cytokines showed lower expression as compared to untreated controls.
IL-12 is an inducer of cell mediated inflammation. In our studies, decreased IL-4 expression in radiation exposed mice was concomitant with increase in IL-12 expression. This antagonistic nature of IL-4 and IL-12 is well reported in literature. When flaxseed was given therapeutically or preventively to irradiated mice, it led to a decrease in IL-12 expression without changing IL-4 status. Flaxseed thus seems to decrease cell mediated inflammation by decreasing IL-12 levels although it does not have any effect on IL-4.
VEGF is identified as an endothelial cell specific growth factor that contributes to angiogenesis and vascular permeability. Radiation exposure to lungs induces hypoxia [42
] and hypoxia itself induces ROS generation which in turn promotes inflammation and vascular damage, activates pro-fibrotic cytokines, and promotes collagen formation. Our results corroborate these reports, as we observed increased VEGF levels in the BAL fluid of radiation-exposed mice, while in FS-fed irradiated animals low VEGF levels were observed as compared to untreated controls.