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

Fig. 1. Experimental plan for assessing NOx (nitrate plus nitrite) supplementation effects on Dox‐treated mice bearing triple‐negative breast cancer (TNBC) tumors, and associated systemic nitrate and nitrite levels in plasma. (A) Following injection of 4T1 cancer cells into BALB/c mice, switching half of the animals after 1 week to drinking water supplemented with sodium nitrate and sodium nitrite, 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). Data are presented as the mean ± standard error of the mean (SEM), with n = 5 and n = 4 for Dox only and Dox + NOx‐treated groups, respectively (see text and Fig. 2 legend for details). Statistical analysis in (B) and (C) was conducted by two‐tailed Student's t‐test. *, P < 0.05, **, P < 0.01.

Fig. 2. Tumor volumes of individual TNBC tumor‐bearing BALB/c mice during Dox treatment in the presence or absence of NOx (nitrate plus nitrite) supplementation. (A) Beginning 13 days after tumor cell injection, animals received four weekly intravenous (IV) doses of doxorubicin (2.5 mg·kg−1 each), and tumor size was measured twice weekly using calipers. Data are presented as the mean ± standard error of the mean (SEM), and statistical analysis was conducted by 2‐way analysis of variance (ANOVA). While the two groups of animals were not significantly different based on the row factor (time since inoculation), they were different based on treatment, with NOx‐supplemented animals overall presenting with smaller tumors (P = 0.0016), and there was no significant interaction between the two factors. (B and C) tumor volumes of individual 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. One animal (which received NOx supplementation) lacked any tumor growth and was excluded from all data shown and used for statistical analysis in this and other figures. Therefore n = 5 for Dox alone, and n =…

Fig. 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 (nitrate plus nitrite) treatment. (C and D) HNE adducts (C) and Prx3 levels (D) were assessed by western blot analysis of frozen LV tissue, indicating statistically significant differences between groups. Data are presented as the mean ± standard error of the mean (SEM), and statistical analysis in (B) through (D) was conducted by two‐tailed Student's t‐test. As in Fig. 2, data from the non‐tumor‐bearing animal was excluded, so n = 5 and n = 4 (biological replicates) for Dox alone or with NOx supplementation, respectively. Multiple technical replicates of each are shown as each analysis was conducted on a separate piece of tissue. In (A) scale bars shown on images (lower right) are 100 μm. *, P < 0.05, **, P < 0.01.

Fig. 4. Effects of NOx (nitrate plus nitrite) 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 NOx‐supplemented animals (P = 0.237). As in the other figures, data from the non‐tumor‐bearing animal was excluded, so n = 5 and n = 4 for Dox alone or with NOx supplementation, respectively. For (A) and (B), scale bars shown on images (lower right) are 100 μm. Statistical analysis in (C) was conducted by two‐tailed Student's t‐test. ns, not significant.