Dietary flaxseed administered post thoracic radiation treatment improves survival and mitigates radiation-induced pneumonopathy in mice

Abstract

Background: Flaxseed (FS) is a dietary supplement known for its antioxidant and anti-inflammatory properties. Radiation exposure of lung tissues occurs either when given therapeutically to treat intrathoracic malignancies or incidentally, such as in the case of exposure from inhaled radioisotopes released after the detonation of a radiological dispersion devise (RDD). Such exposure is associated with pulmonary inflammation, oxidative tissue damage and irreversible lung fibrosis. We previously reported that dietary FS prevents pneumonopathy in a rodent model of thoracic X-ray radiation therapy (XRT). However, flaxseed's therapeutic usefulness in mitigating radiation effects post-exposure has never been evaluated.

Methods: We evaluated the effects of a 10%FS or isocaloric control diet given to mice (C57/BL6) in 2 separate experiments (n = 15-25 mice/group) on 0, 2, 4, 6 weeks post a single dose 13.5 Gy thoracic XRT and compared it to an established radiation-protective diet given preventively, starting at 3 weeks prior to XRT. Lungs were evaluated four months post-XRT for blood oxygenation levels, inflammation and fibrosis.

Results: Irradiated mice fed a 0%FS diet had a 4-month survival rate of 40% as compared to 70-88% survival in irradiated FS-fed mouse groups. Additionally, all irradiated FS-fed mice had decreased fibrosis compared to those fed 0%FS. Lung OH-Proline content ranged from 96.5 ± 7.1 to 110.2 ± 7.7 μg/ml (Mean ± SEM) in all irradiated FS-fed mouse groups, as compared to 138 ± 10.8 μg/ml for mice on 0%FS. Concomitantly, bronchoalveolar lavage (BAL) protein and weight loss associated with radiation cachexia was significantly decreased in all FS-fed groups. Inflammatory cell influx to lungs also decreased significantly except when FS diet was delayed by 4 and 6 weeks post XRT. All FS-fed mice (irradiated or not), maintained a higher blood oxygenation level as compared to mice on 0%FS. Similarly, multiplex cytokine analysis in the BAL fluid revealed a significant decrease of specific inflammatory cytokines in FS-fed mice.

Conclusions: Dietary FS given post-XRT mitigates radiation effects by decreasing pulmonary fibrosis, inflammation, cytokine secretion and lung damage while enhancing mouse survival. Dietary supplementation of FS may be a useful adjuvant treatment mitigating adverse effects of radiation in individuals exposed to inhaled radioisotopes or incidental radiation.

Figure 1

Experimental Plan. Mice were fed with 0% or 10%FS diet initiated prior (-3 weeks) or post (+2, +4, +6 weeks) single fraction (13.5 Gy) X-ray radiation therapy (XRT). Mice were sacrificed at 4 months post-XRT.

Figure 2

Detection of flaxseed lignan metabolites in blood. Circulating lignan (ED and EL) levels in plasma of mice, 4 months post-XRT, were determined using GC/MS/MS. Mice were fed with 0% or 10%FS diet initiated prior (-3 weeks) or post (+2, +4, +6 weeks) X-ray radiation therapy (XRT). Data is represented mean ± SEM (n = 3 mice per group).

Figure 3

Effect of Flaxseed (FS) diet on body weight of mice 4 months post-XRT. Mice were fed with 0% or 10%FS diet prior (-3 weeks) or post (+2, +4, +6 weeks) X-ray radiation therapy (XRT). Body weight was recorded at 4 months post-irradiation. Data is represented mean ± SEM of two independent experiments (n = 15-25 mice per group). The white, black, gray and hatched bars represent untreated control, 0%FS + XRT, 10%FS and 10%FS + XRT groups, respectively. The dotted line represents weight average in non-irradiated control animals. *p ≤ 0.01 for irradiated 0%FS vs. irradiated 10%FS, ^$p ≤0.01 for irradiated vs. non-irradiated 0% and #p ≤ 0.05 irradiated 10%FS (initiated 3 weeks prior to XRT) vs. all irradiated 10%FS (diet initiated on, or post-XRT).

Figure 4

Effect of Flaxseed (FS) diet on the survival of mice 4 months post-XRT. Mice were fed with 0% or 10%FS diet prior (-3 weeks) or post (+2, +4, +6 weeks) X-ray radiation therapy (XRT) and observed for survival up to 16 weeks post-irradiation. Data is represented mean ± SEM of two independent experiments (n = 15 and 25 mice in non-irradiated and irradiated groups, respectively). Panel A: Kinetics of mouse survival. Panel B: Mouse survival 4 months post-XRT. The white, black, gray and hatched bars represent untreated control, 0%FS + XRT, 10%FS and 10%FS + XRT groups, respectively.

Figure 5

Evaluation of Lung Injury, Inflammation and Blood Oxygenation Levels in mice 4 months post-XRT. Mice were fed with 0% or 10%FS diet at designated times (-3, 0, +2, +4, +6 weeks) of X-ray radiation therapy (XRT). Data is represented mean ± SEM of two independent experiments (n = 15-25 mice per group). Panel A: Total WBC counts in bronchoalveolar lavage (BAL) of irradiated (13.5 Gy, XRT) mice after 4 months. *p ≤ 0.05 for irradiated 0%FS vs. irradiated 10%FS Panel B: Total proteins in BAL of mice after 4 months. *p ≤ 0.01 for irradiated 0%FS vs. irradiated 10%FS Panel C: Arterial O₂ levels were measured using pulse oximetry in irradiated (13.5 Gy, XRT) mice after 4 months. The white, black, gray and hatched bars represent untreated control, 0%FS + XRT, 10%FS and 10%FS + XRT groups, respectively. *p ≤ 0.05 for irradiated 0%FS vs. irradiated 10%FS.

Figure 6

Histological evaluation of lung Hematoxylin and Eosin (H&E)-stained sections post-XRT (4 months). Mice were fed with 0% or 10%FS diet prior (-3 weeks) or post (+2, +4, +6 weeks) X-ray radiation treatment (XRT). Lungs were harvested 4 months post single fraction XRT and processed for histology. Asterisks designate proteinaceous exudate in alveolar spaces.

Figure 7

Effect of 10%FS diet on lung lipid peroxidation levels 4 months post-XRT. Mice were fed with 0% or 10%FS diet prior (-3 weeks) or post (+2, +4, +6 weeks) X-ray radiation therapy (XRT). Lungs were harvested after 4 months, homogenized and assayed for lipid peroxidation by measuring Malondialdehyde (MDA) levels. MDA is calculated per g of wet lung tissue. Data is represented mean ± SEM of two independent experiments (n = 15-25 mice per group). The white, black, gray and hatched bars represent untreated control, 0%FS + XRT, 10%FS and 10%FS + XRT groups, respectively. *p ≤ 0.0002 for irradiated 0%FS vs. irradiated 10%FS.

Figure 8

Determination of fibrotic changes in mouse lungs 4 months post-XRT. Panel A: Evaluation of OH-Proline content in lungs of irradiated (13.5 Gy, XRT) mice. Mice were fed with 0% or 10%FS diet prior (-3 weeks) or post (+2, +4, +6 weeks) XRT. Lungs were harvested after 4 months and OH-Proline assay was performed. *p ≤ 0.005 for irradiated 0%FS vs. irradiated 10%FS. Panel B: Fibrotic Index range = 0 - 4). Data is represented mean ± SEM of two independent experiments (n = 15-25 mice per group). The white, black, gray and hatched bars represent untreated control, 0%FS + XRT, 10%FS and 10%FS + XRT groups respectively. *p ≤ 0.005 for irradiated 0%FS vs. irradiated 10%FS. *p ≤ 0.005 for irradiated 0%FS vs. irradiated 10%FS.

Figure 9

Histological evaluation of lung Trichrome-stained sections post-XRT (4 months). Mice were fed with 0% or 10%FS diet prior (-3 weeks) or post (+2, +4, +6 weeks) XRT. Lungs were harvested 4 months post single fraction XRT and processed for histology. Arrows designate collagen deposition in lung (blue color).