Background
Ionizing radiation can cause deleterious effects in living organisms. Technological advancement has increased human exposure to ionizing radiation through diagnostic and therapeutic radiographic procedures, as well as through daily workplace activities [
1]. Humans are also exposed to ionizing radiation above background levels during air and space travel, from nuclear accidents, and through the use of electronic devices. Additionally, global developments over the past decade have established terrorism as a novel and highly concerning means by which large numbers of people could be exposed to potentially lethal amounts of radiation [
2].
There are at least two potential ways that a terroristic attack could expose a population to radiation injury. If terrorists gained possession of a nuclear warhead, detonation could release large amounts of radiation (in a single “blast”) that could induce radiation sickness, bone marrow damage, and potential lung injury. More likely, the weapon of radiological terrorism would be a “dirty bomb,” or a radiological dispersion device (RDD). Conventional explosives would spread radioactive materials in the form of powder or pellets [
2‐
4] that could spread far away from the immediate explosion and pose a significant health risk if inhaled. Whole-body irradiation induces acute radiation syndrome (ARS) with symptoms caused by damage to the hematopoietic, gastrointestinal and central nervous systems [
5]. The lung becomes the target organ for radiation injury from an RDD.
Radiation pneumonopathy is defined as a significant clinical toxicity from thoracic radiation [
6,
7]. Patients receiving large doses of radiation to the lung demonstrate two adverse clinical scenarios [
8]. An acute type of toxic radiation response can occur within weeks after irradiation followed by a second type of radiation-induced lung injury which can begin within several months after exposure; This is characterized as the “late fibrotic” phase, in which the number of inflammatory cells (particularly neutrophils) decrease and a marked thickening of alveolar walls due to collagen deposition can be noted histopathologically [
9,
10]. Radiation pneumonopathy has been modeled in animals [
9]; C57/BL6 mice are especially susceptible to this fibrotic reaction [
11‐
13].
Several agents, ranging from cytokines to receptor blockers, have been tested for their efficacy in ameliorating radiation effects [
12,
14‐
16]. Most agents, even those proven to be effective as radioprotectors (administered prior to a radiation exposure) unfortunately are not yet available for human use. These agents were intended as treatments for radiation injuries resulting from the therapeutic use of radiation — a very different scenario compared to radiation injuries resulting from nuclear accidents or radiological terrorism. In most accidental or terrorism scenarios 1) treatment would not be initiated until after the irradiation, thus eliminating agents that work only when given before irradiation; 2) radiation would be received in a short time frame and agents effective in multi-week radiation treatments might be less effective for a single large dose of radiation; and 3) a mitigator would need to be administered to a large population of healthy individuals exposed to an undetermined dose of radiation. Therefore, it has become highly desirable to find an agent that is non-toxic, cost-effective, and safe for multiple administrations with beneficial effects spanning a long radiation exposure and post-radiation exposure recovery phase.
Significant systemic toxicity associated with chemical radioprotectors [
17,
18] has shifted the focus to plants, herbs, as well as antioxidant agents to evaluate their radioprotective potential [
19]. Our group has identified flaxseed (FS) and its bioactive lignan component (FLC) as potent protectors against radiation-induced lung injury when given prior to radiation exposure [
20‐
22]. Specifically, dietary FS decreased radiation-induced oxidative lung tissue damage, decreased lung inflammation and prevented lung fibrosis. Our previous work demonstrated that FS given after thoracic radiation mitigated radiation effects by decreasing cytokine release, inflammation, and pulmonary fibrosis while improving mouse survival [
22]. We also recently showed that FS has also potent radiation mitigating properties [
22]. Our study of the whole grain, however, did not allow for the identification of the bioactive ingredient of FS that mediated radioprotective- and radiation-mitigating properties. We further designed studies that provided the first evidence that FLC, the lignan component in FS enriched in the phenolic, secoisolariciresinol diglucoside (SDG) surpassed whole grain FS in terms of antioxidant, anti-inflammatory and anti-fibrotic properties [
20] and was indeed responsible for the radioprotective properties of the whole grain. However, the radiation mitigating effects of FLC (and the lignan SDG more specifically) were never investigated. The current study was performed to ascertain whether FLC, in addition to its radioprotective properties, could also be an effective mitigator of radiation toxicity when administered at different time points soon after radiation exposure to the lung. Evidence provided in this study provides novel, strong support that the bioactive ingredient in whole grain FS responsible for its radiation mitigating properties is the lignan component and more specifically SDG. Focusing on SDG as a radiation mitigator will allow detailed mechanistic studies in the future and further development into a drug with clinical usefulness thus showing how from a natural product and a common botanical, a chemical agent can be identified with enormous clinical implications.
Discussion
We have shown here for the first time that the lignan component of flaxseed, enriched in the lignan SDG, administered within just 24–72 hours post-thoracic radiation enhanced survival and mitigated the chronic lung injury induced by XRT. We determined that FLC-supplemented diet mitigated the deleterious effects of XRT by: 1) improving blood oxygenation levels, 2) decreasing lung injury by lowering BAL protein levels, 3) reducing pulmonary fibrosis by decreasing collagen content and TGF-beta1 levels of lung tissues, 4) reducing lung inflammation by decreasing WBC influx into the airways and most importantly, 5) reducing oxidative tissue damage as shown by decreased protein nitration in lung tissue and lipid peroxidation in BAL fluid 6) improving overall animal survival.
Radiation-induced injury to adjacent normal tissue is a notable sequelae of ionizing radiation exposure [
7]. Our results indicated that XRT-induced lung inflammation and impaired blood oxygenation (decreased SaO2) were improved with 10 and 20% FLC diet when initiated after XRT. Vujaskovic and coworkers [
7,
34] have shown that severe hypoxia develops months post an initial radiation exposure of lung tissues. Such hypoxia resulted from the development of a cascade of events leading to lung injury. Improved blood oxygenation of all FLC-fed mouse groups may lead to decreased levels of tissue hypoxia and may therefore explain the mitigation of adverse radiation effects even when diets are initiated post challenge. Decrease of radiation-induced lung inflammation by a mitigator was first shown by our group in whole grain FS [
22]. This is the first report that FLC mitigated pulmonary inflammation when given hours post initial XRT challenge.
A major feature of radiation pneumonitis is a considerable increase in alveolar protein accumulation, an indicator of increased vascular permeability and direct lung injury [
35]. Radiation damages resident lung cells that subsequently release inflammatory cytokines and chemokines that recruit inflammatory cells to that area of injury while priming the immune system in a cyclic-feedback loop [
36]. BAL protein levels are a direct and reliable measure of lung injury, translating into actual tissue damage while lung inflammatory cell markers (although useful) serve as a surrogate measure of inflammation and not injury directly [
34]. Our results show significant mitigation of lung injury in all the experimental FLC diet fed groups, regardless of the timing of diet initiation. This may be attributed to decreased inflammatory cell influx and membrane oxidation in FLC-supplemented mice. In addition, we noted decreased oxidative tissue damage in FLC-fed mice, as evidenced by the tissue levels of nitrotyrosine and MDA levels in the BAL fluid. Antioxidant agents capable of decreasing nitrotyrosine have also been shown by others to be protective in radiation lung damage [
37]. We first reported [
22] that whole grain FS was able to mitigate physiological lung injury from radiation
in vivo. We now are the first, to our knowledge, to report that FLC demonstrates similar
in vivo beneficial properties as the whole grain, strongly suggesting that FLC contains the key bioactive radiation mitigator found in FS.
Radiation pneumonitis chronically evolves into fibrotic changes within lung tissues as a late phase of the radiation exposure response mechanism [
38]. We have shown that whole grain FS was protective against experimental radiation fibrosis [
21] and as a mitigator of late radiation induced lung changes after one dose of thoracic XRT [
22]. Our current study showed for the first time that via FLC, fibrotic processes associated with high TGF-beta1 levels in lung tissues can be blunted even when the protective agent is given post-radiation damage, i.e., as a radiation mitigator. Notable benefits including quantitative and qualitative physiologic and histopathologic endpoints from the therapeutic use of FLC diet when initiated at 24, 48, 72 hours posts XRT demonstrate that FLC is a highly bioactive radiation mitigator. However, FLC-mediated decline in both lung hydroxyproline levels and fibrotic index were more prominent when diet was started preventively, i.e., 3 weeks prior to XRT. This suggests that further development or modification of the bioactive component(s) of FLC has the potential to further improve the properties of this novel dietary radiation mitigator.
Regulatory cytokines and chemokines play a significant role in the inflammatory response implicated in radiation induced lung injury. The detection of pro-inflammatory cytokines, such as IL-6 and IL-12, in the BAL of irradiated mice at 4 months post-XRT signifies a chronic and sustained inflammatory state that occurs post-irradiation. This heightened level of inflammation may play a significant role in the development of pulmonary fibrosis. We have previously reported that whole grain flaxseed when given as radiation mitigator reduces the concentration of pro-inflammatory cytokines present in BAL fluid [
22]. Based on our current findings, this reduction in BAL cytokine levels may be conferred by the lignan component in whole grain flaxseed.
Lung lavage has been advocated as a possible countermeasure to remove radionuclides inhaled after detonation of an RDD [
39,
40]. However, this seems impractical since multiple lavages may be required for efficient removal of radionuclide burden using an invasive procedure with inherent risks and a requirement of anesthesia. Alternatively, a plethora of synthetic and natural compounds are being evaluated with the intent of mitigating radiation damage [
19]. Agents are being developed that target inflammatory cell recruitment, free radical production, cell death, cytokine and growth factor expression, and other cell functions [
5,
10,
41]. While Amifostine is the only FDA approved cytoprotective radiation mitigator its use has been associated with significant systemic toxicity [
19]. Furthermore, many of the compounds that had offered cellular radioprotection
in vitro have not demonstrated pre-clinical
in vivo efficacy, as summarized in a recent review by Williams et.al. [
10]. Despite much progress, the search for an effective and safe radiation mitigator with clinical usefulness has yet to be identified.
An ideal radiation mitigator should be safe, effective, have an easy route of administration and a long shelf-life. The bioactive flaxseed lignan complex (FLC) enriched in the phenolic secoisolariciresinol diglucoside (SDG) has become a topic of study because of its anti-inflammatory, antioxidant and anti-fibrotic attributes most notably in models of thoracic radiation induced acute and chronic lung injury [
20]. Importantly, prolonged FLC administration in our animal models has not led to any significant toxicity. This is a critically important feature, since administering a radiation mitigator that possessed even mild toxicity in healthy individuals to a large population following radiation exposure could lead to severe toxicity in individuals with additional medical co-morbidities. Furthermore, FLC has a long, stable shelf life and is easy to administer orally at an affordable price. We therefore posit that FLC may be an effective, safe and cost-effective mitigator of radiation damage.
It is evident from our results that FLC diet given within 24–72 hours after thoracic radiation exposure has benefits in terms of morbidity and mortality. Improvement of survival using antioxidants such as N-acetyl-Cysteine (NAC) or mitochondrial-targeted small molecule radiation damage mitigators have been shown in mouse models of abdominal irradiation [
42] or total body irradiation [
43] respectively. To our knowledge our study is the first to report that FLC served this same purpose improving survival of animals in an experimental model of thoracic radiation damage without any significant side effects or difficulties in oral administration, often associated with NAC given the unpleasant smell of its oral formulation.
In summary, we have evaluated a non-toxic, widely available dietary phenolic compound that yielded late protective benefits after lung exposure to radiation. We have studied the beneficial properties of whole grain FS in the past. Here for the first time we demonstrated that FLC surpassed whole grain FS in its antioxidant, anti-inflammatory and anti-fibrotic mitigation properties when administered after thoracic XRT. FLC altered immediate XRT-induced markers of lung damage, creating a radioprotective milieu post-XRT, and provided high levels of circulating antioxidants from the continued metabolism of its bioactive lignans. Our long-term goal is to provide that long awaited, safe to consume, easy to administer, inexpensive compound for large populations in either the post-therapeutic radiation scenario or post-radiologic terrorism (dirty-bomb) event. That compound is the flaxseed lignan complex.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MCS: Designed the study and individual experiments, analyzed data, wrote the manuscript and supervised project and lab personnel; JBT: Analyzed data, assisted in writing of the manuscript, and performed final editing; ST: Assisted with animal experiments and manuscript preparation; SH: performed all the irradiation procedures; RP: Performed animal experiments, biochemical assays and conducted data analysis; FD: Assisted with pulse oximetry; EA: Conducted animal experiments and tissue analyses; CCS: Performed pathology assessment of histological specimens; KAC: Provided consultation on data analysis; TB and SGC assisted with the TGF- beta evaluation in tissues. All authors read and approved the final manuscript.