Dietary flaxseed prevents radiation-induced oxidative lung damage, inflammation and fibrosis in a mouse model of thoracic radiation injury

Abstract

Flaxseed (FS) has high contents of omega-3 fatty acids and lignans with antioxidant properties. Its use in preventing thoracic X-ray radiation therapy (XRT)-induced pneumonopathy has never been evaluated. We evaluated FS supplementation given to mice given before and post-XRT. FS-derived lignans, known for their direct antioxidant properties, were evaluated in abrogating ROS generation in cultured endothelial cells following gamma radiation exposure. Mice were fed 10% FS or isocaloric control diet for three weeks and given 13.5 Gy thoracic XRT. Lungs were evaluated at 24 hours for markers of radiation-induced injury, three weeks for acute lung damage (lipid peroxidation, lung edema and inflammation), and at four months for late lung damage (inflammation and fibrosis). FS-Lignans blunted ROS generation in vitro, resulting from radiation in a dose-dependent manner. FS-fed mice had reduced expression of lung injury biomarkers (Bax, p21 and TGF-beta1) at 24 hours following XRT and reduced oxidative lung damage as measured by malondialdehyde (MDA) levels at 3 weeks following XRT. In addition, FS-fed mice had decreased lung fibrosis as determined by hydroxyproline content and decreased inflammatory cell influx into lungs at 4 months post XRT. Importantly, when Lewis lung carcinoma cells were injected systemically in mice, FS dietary supplementation did not appear to protect lung tumors from responding to thoracic XRT. Dietary FS is protective against pulmonary fibrosis, inflammation and oxidative lung damage in a murine model. Moreover, in this model, tumor radioprotection was not observed. FS lignans exhibited potent radiation-induced ROS scavenging action. Taken together, these data suggest that dietary flaxseed may be clinically useful as an agent to increase the therapeutic index of thoracic XRT by increasing the radiation tolerance of lung tissues.

Figure 1. Single fraction thoracic XRT treatment

Panels A-C: Mice are anesthetized mildly and irradiated in a custom-made immobilization chamber that allows exposure of the lungs (bilateral) with shielding of the head, abdomen, and extremities (A,B). The dose of irradiation is a single fraction delivered via single AP (anterior-posterior) approach. The dose used is 13.5 Gy (roughly corresponding to LD50). For quality assurance, theromoluminescent dosimeters are placed over selected mice, to verify correct dose administration. Hair loss and discoloration designates and confirms the site or irradiation.

Figure 2. Antioxidant action of Flaxseed in irradiated lung cells

Panel A: ROS generation was measured in mouse PMVEC treated with SDG shortly prior to 2 Gy gamma irradiation. Fluorescence intensity was measured in the absence of SDG or treatment with 0.1−25 μM SDG. The ROS-specific dye H₂DCF-DA was used to monitor ROS generation. Panel B: 8 random fields were selected for the quantification of fluorescence (B). *P<0.001 for irradiated control as compared to all doses of SDG given to gamma-irradiated cells shortly prior to irradiation.

Figure 3. Antioxidant action of Flaxseed in irradiated lung tissues

Malondialdehyde (MDA) levels were evaluated in whole mouse lung homogenates from non-irradiated, baseline controls and compared to control or 10% Flaxseed fed mice at three weeks after thoracic XRT. Values represent mean ± SEM. n=10−15 mice per group. *p=0.001 for 0% FS vs. 10% Flaxseed + XRT and **p=0.04 for irradiated 0% FS vs. non-irradiated 0% FS.

Figure 4. Flaxseed modulates post XRT gene expression involved with apoptosis, cell cycle regulation, and inflammation

RTPCR of Bax, p21, and TGFβ-1 at 24 hours post XRT in mice fed control or 10% Flaxseed diets. Values represent means of triplicate samples of pooled cDNA from n=3 mice per group. Values were normalized to beta actin gene levels and compared to unirradiated controls. Error bars represent mean ± SEM. *p<0.001 vs. 0% Flaxseed at 24h post XRT for Bax, p21, and TGF-β1.

Figure 5. Evaluation of FS in abrogating XRT-induced inflammation

Mice we re fed a 0% or 10% FS-supplemented diet for 3 weeks and then exposed to a single fraction thoracic XRT (13.5 Gy). Mice were kept on the respective diets and sacrificed after 4 months (Late phase) for evaluation of lung inflammation and injury. Panels A,B: Comparison of alveolar PMN and Macrophage influx in lungs 4 months post-XRT. Cells were quantified from histological H&E-stained lung sections in 5 high-power fields per lung in all animals from 3 separate long-term studies. *p<0.0001 for 0% FS vs. 10% FS XRT and **p<0.0001 vs. 0% FS no XRT in the late phase.

Figure 6. Lung fibrosis four months after thoracic irradiation

Panel A: Evaluation of collagen deposition using Mason's trichrome blue stain (MTB) in lungs from mice fed control or 10% FS diets at four months ± thoracic XRT. Characteristic morphology of each group is shown at 100x (Panel a,c) and at higher magnification at 400x (Panels b, d). Panel B: Semi-quantitative assessment of lung fibrosis using a radiation Fibrotic Index (FI) generated by a lung pathologist, with 4 signifying maximal fibrosis. Values represent mean ± SEM of n=5−14 slides per group, **p=0.008 vs. 0% FS, No XRT and *p=0.005 vs.10% + XRT. Panel C: Evaluation of hydroxyproline content in mice treated as in A and B. Values indicate mean ± SEM of n=10 mice per group. (**p=0.0004 vs. 0% FS, No XRT and *p=0.002).

Figure 7. Dietary Flaxseed treatment and lung tumor protection

Panel A: Mice were injected with 2x106 LLC cells or saline and lung tumors were established within 10 days post injection. Twenty five days prior to a single dose of XRT (13.5 Gy) to the thorax at T=0, the mice were placed on a 0% or 10% Flaxseed diet. Mice were allowed to live 4 additional weeks and lungs were excised and weighed. (n=4 mice/group). Panel B: Lung weight evaluations of 0% Flaxseed as compared to 10% flaxseed supplemented. The data represent Mean ± SEM. *p=0.04 vs. Lung + tumor, no XRT vs. plus XRT. Dashed line represents non-irradiated, non-treated, control mouse lungs at initiation of the experiments. Panel C: Morphometric analysis of % tumor in total area per treatment group, Mean ± SEM *p=0.005 for 0% FS tumor/no XRT vs. 0% FS Tumor+ XRT.