Dietary nitrate and nitrite protect against doxorubicin-induced cardiac fibrosis and oxidative protein damage in tumor-bearing mice

Figure 1.. Experimental plan for assessing NOx supplementation effects on Dox-treated mice bearing triple negative breast cancer (TNBC) tumors, and associated nitrate and nitrite levels in plasma. A, Following injection of 4T1 cells and growth of tumors for 13 days, echocardiography (ECHO) was performed, then mice were treated with four weekly doses of Dox, again evaluated by ECHO, then sacrificed to harvest tissues for analysis. B and C, Plasma was evaluated for concentrations of nitrite (B) and nitrate (C). *, p<0.05, ***, p<0.001

Figure 2.. Tumor volumes of individual TNBC tumor-bearing BALB/c mice during Dox treatment in the presence or absence of NOx supplementation. A, Beginning 13 days after tumor cell injection, animals received four weekly intravenous (IV) doses of doxorubicin (2.5 mg/kg each), and tumor size was measured twice weekly using calipers. B and C, tumor volumes of mice treated with Dox alone (B), or Dox in combination with Nox-supplemented H2O (C), were compared at their maximal size and at the endpoint of 41 days.

Figure 3.. Protection of Dox-treated cardiac tissue from fibrosis and formation of protein adducts with 4-hydroxynonenal (HNE) by nitrate and nitrite supplementation. A, Fibrosis was evaluated using PicroSirius Red staining of left ventricle (LV) cross-sections. B, Fibrosis was significantly decreased with NOx treatment. C and D, HNE adducts (C), and Prx levels (D), were assessed by Western blot analysis with frozen LV tissue, indicating statistically-significant differences between groups. *, p<0.05, ***, p<0.001, ****, p<0.0001

Figure 4.. Effects of NOx supplementation on lung tissues and TNBC-associated metastatic lesions. A and B, Hematoxylin and Eosin (H&E) stained lung tissue from Dox-exposed mice showed significant evidence of tissue remodeling (A) which was less evident in the tissue from the NOx-supplemented mice (B). C, numbers of metastatic lesions in lung tissue associated with TNBC and Dox treatment were similar but trended lower in the NOxsupplemented animals (p=0.237). ns, not significant