Effect of 3,3'-Diindolylmethane on Pulmonary Injury Following Thoracic Irradiation in CBA Mice

Abstract

The molecule 3,3'-diindolylmethane (DIM) is small, a major bioactive metabolite of indole-3 carbinol (13C), and a phytochemical compound from cruciferous vegetables released upon exposure to the gut acid environment. DIM is a proposed anti-cancer agent and was previously demonstrated to prevent radiation damage in the bone marrow and the gastrointestinal tract. Here we investigated the effect of DIM on radiation-induced injury to the lung in a murine model through untargeted metabolomics and gene expression studies of select genes. CBA mice were exposed to thoracic irradiation (17.5 Gy). Mice were treated with vehicle or DIM (250 mg kg, subcutaneous injection) on days -1 pre-irradiation through +14 post-irradiation. DIM induced a significant improvement in survival by day 150 post-irradiation. Fibrosis-related gene expression and metabolomics were examined using lung tissue from days 15, 45, 60, 90, and 120 post-irradiation. Our qRT-PCR experiments showed that DIM treatment reduced radiation-induced late expression of collagen Iα and the cell cycle checkpoint proteins p21/waf1 (CDKN1A) and p16ink (CDKN2A). Metabolomic studies of lung tissue demonstrated a significant dampening of radiation-induced changes following DIM treatment. Metabolites associated with pro-inflammatory responses and increased oxidative stress, such as fatty acids, were suppressed by DIM treatment compared to irradiated samples. Together these data suggest that DIM reduces radiation-induced sequelae in the lung.

Figure 1:

Experimental set-up for DIM administration and thoracic irradiation. CBA mice, 12-14 weeks of age, were exposed to 17.5 Gy thoracic irradiation or sham irradiated (sham+vehicle). Irradiated (Rad) mice either received vehicle or DIM by intraperitoneal injection from days −1 pre-irradiation through day +14 post-irradiation.

Figure 2:

Kaplan-Meier curves and animal weights to evaluate the effects of DIM following thoracic irradiation. CBA mice, 12-14 weeks of age, were exposed to 17.5 Gy thoracic irradiation or sham irradiated. Irradiated and sham irradiated mice either received vehicle (Sham+Vehicle or Rad+Vehicle) or DIM (Sham+DIM or Rad+DIM) by intraperitoneal injection from days −1 pre-irradiation through day +14 post-irradiation as described in the Methods. A. Kaplan-Meier survival curves. For both irradiated groups N = 18 for the survival studies. Graph shows data from two independent experiments. * indicates statistical significance by Log rank test and by Gehan-Breslow-Wilcoxon test compared to Rad+Vehicle; for both analyses, p<0.05. B. Average weights of animals. Weights for animals were obtained pre-irradiation (negative time points) and post-irradiation. Data show means ± SEM, N= 3-8, with fewer animals at the 120 day time points. * indicates statistical significance from sham+vehicle at the same time point (p<0.05).

Figure 3:

Lung histology at 120 days post-irradiation. CBA mice, 12-14 weeks of age, were exposed to 17.5 Gy thoracic irradiation or sham irradiated. Irradiated and sham irradiated mice either received vehicle (Sham+Vehicle or Rad+Vehicle) or DIM (Sham+DIM or Rad+DIM) by intraperitoneal injection from days −1 pre-irradiation through day +14 post-irradiation as described in the Methods. A. Sham+Vehicle; B. Sham+DIM; C. Rad+Vehicle; D. Sham+DIM. 20X magnification is shown.

Figure 4:

Heatmaps at days 15 and 120 post-irradiation. Heatmaps with Euclidean distance and Ward clustering algorithm represent the top 100 ions through ANOVA for each analysis.

Figure 5:

Representative metabolites over the course of 120 days post-irradiation. All data are represented as mean ± standard errors of the mean (SEM) with N for each group shown in Supplementary Table 1. * represents a p-value of <0.05 determined through a t-test with 10 % FDR correction.

Figure 6:

Effect of DIM on radiation-induced changes in gene expression in the lung. CBA mice, 12-14 weeks of age, were exposed to 17.5 Gy thoracic irradiation (Rad). Rad mice received either vehicle or DIM by intraperitoneal injection from days −1 pre-irradiation through day +14 post-irradiation. Mice were euthanized at the indicated times and lung tissue was obtained for RNA purification. RT-PCR was performed for A. Colla1; B. MMP9; C. p21; and D. p16. Data show means +/− SD from N = 4-5 animals/group. * indicates significance from Radiation + vehicle at the same time point.