DOI: 10.5061/dryad.ghx3ffc1p
title: Dietary nitrate and nitrite protect against doxorubicin-induced cardiac fibrosis and oxidative protein damage in tumor-bearing mice

description: Anthracycline-induced cardiotoxicity remains a major limitation in cancer
 therapy, affecting long-term cardiovascular health in survivors. Dietary
 nitrate supplementation has shown cardioprotective effects in preclinical
 models of doxorubicin (Dox)-induced and ischemia-reperfusion injury, but
 it is unclear whether nitrate and/or nitrite (NOx) would have adverse
 effects on the anti-cancer efficacy of the drug. To evaluate Dox efficacy
 against triple negative breast cancer (TNBC) in the presence of dietary
 nitrate and nitrite, tumor-bearing BALB/c mice (N=5 mice per group, 10
 mice total) were treated with 4 weekly intravenous doses of Dox with or
 without NOx supplementation of their drinking water. Cardiac tissue from
 the NOx-treated mice exhibited less fibrosis and lower levels of
 4-hydroxynonenal-modified proteins, a marker of lipid oxidation and
 oxidative stress. Tumor sizes varied, but most regressed by the final Dox
 dose. Importantly, NOx supplementation did not compromise the anti-tumor
 efficacy of Dox nor did it promote pulmonary metastasis; instead, a trend
 toward fewer metastatic lesions was observed. These findings support the
 potential clinical use of dietary nitrate and nitrite as adjuncts to Dox
 treatment to mitigate cardiotoxicity without impairing anti-cancer
 outcomes.
description: # Dietary nitrate and nitrite protect against doxorubicin-induced cardiac
 fibrosis and oxidative protein damage in tumor-bearing mice Dataset DOI:
 [10.5061/dryad.ghx3ffc1p](10.5061/dryad.ghx3ffc1p) All of these datasets
 reflect data collected from the 10 study mice (all with breast cancer
 tumors and doxorubicin treatment, half with nitrite and nitrate
 supplementation of their drinking water), either during treatment, or
 after sacrifice.  Data include echocardiography outputs for all mice
 before and after the treatments as well as measurements of tumor sizes
 across time of the study, and outcomes of tissue samples collected after
 sacrifice.  Nitrite and nitrate levels in plasma were analyzed by
 chemiluminescence, tissue staining and imaging was used to measure cardiac
 tissue fibrosis and lung tissue metastatic lesions, and cardiac tissue was
 analyzed by Western blot to evaluate HNE (hydroxynonenal) adducts and Prx3
 (peroxiredoxin 3) levels. ## Description of the data and file structure
 This study involved inoculation of 10 mice with breast cancer cells,
 growth of tumors, then treatment with 4 weekly intravenous doses of
 Doxorubicin (Dox), with echocardiography conducted before the first and
 after the last Dox treatments, then sacrificing of the mice for tissue
 collection and analysis. Female 6-week-old BALB/c mice (n = 10) were
 purchased from Jackson Laboratory and put on a low nitrate diet (Harlan
 Teklad TD 99366) one day post arrival.  One week later mice were each
 injected with syngeneic 4T1 triple negative breast cancer (TNBC) cells (1
 × 10^6^ cells) in the left fourth mammary gland fat pad to induce tumors.
 Mice were randomly divided into two groups: the first (control) group
 received Dox therapy, and the second (test) group was continuously
 provided with both sodium nitrate (1 g/L) and sodium nitrite (0.9 g/L) in
 their drinking water (replaced weekly) in addition to the Dox therapy.
 Once the tumor volume reached about 50 mm^3^, 2.5 mg/kg of
 intravenously-administered doxorubicin was injected into the tail vein
 once weekly for 4 weeks to reach an accumulated dose of 10 mg/kg. Tumor
 volumes were measured twice per week and echocardiography was performed
 before the first and after the last Dox treatment. At the study endpoint
 one week after the final dose of Dox, mice were humanely euthanized and
 plasma, tumors, lungs, and heart were harvested for analysis. One mouse
 that received NOx supplementation did not develop a tumor, therefore in
 all except the Echocardiography data, the tumor-free mouse data were
 excluded. ### Files and variables Description: Data were collected from
 mice and their harvested tissues after all were treated with Dox, but half
 (N=5) received regular drinking water whereas the other half (N=5)
 received nitrate and nitrite (together termed “NOx”) in their drinking
 water.  Datafiles * Yammani-Nitrite_and_nitrate_levels_in_plasma.csv *
 Yammani-Tumor_volumes.csv * Yammani-Cardiac_Tissue_HNE_Adducts.csv *
 Yammani-Cardiac_Tissue_Prx3_levels_.csv *
 Yammani-Lung_Tissue_Metastatic_Lesions.csv *
 Yammani-Cardiac_Tissue_Fibrosis.csv * Yammani-Echocardiography_data.csv
 1\. Nitrate and Nitrite Levels in Plasma Plasma obtained at the time of
 sacrificed after centrifugation was stored at -80 oC, then analyzed by
 chemiluminescence for plasma nitrate (NO~3~^-^) and nitrite (NO~2~^-^)
 levels (collectively referred to as “NOx”) using an HPLC-based Eicom NOx
 Analyzer, model ENO-20.   Data indicate plasma concentrations of each
 analyte in µM for the two groups, “Dox only” or “Dox+NOx” with n=5 and n=4
 for Dox only and Dox + NOx treated groups, respectively, after removing
 the tumor-free mouse data. 2\. Tumor Volumes Tumor sizes were measured
 twice weekly using calipers. Tumor volumes were obtained from measurements
 of the longest perpendicular axes: ((long axis) x (short axis)^2)/2).
   Volume values are reported in cubic mm. 3\. Cardiac Tissue Fibrosis
 Cardiac fibrosis of left ventricle (LV) cross-sections was evaluated using
 PicroSirius Red staining to detect collagen and image analysis software
 (ImageJ). 4\. Cardiac Tissue HNE Adducts Cardiac tissue samples rapidly
 frozen in liquid nitrogen were used to conduct Western blotting for
 4-Hydroxynonenal (HNE) adduct formation on proteins with an antibody from
 Abcam (ab46545).  Densitometry was used to evaluate band intensity. 5\.
 Cardiac Tissue Prx3 Levels Cardiac tissue samples rapidly frozen in liquid
 nitrogen were used to conduct Western blotting for peroxiredoxin-3 (Prx3)
 levels with an antibody from Abcam (ab73349).  Densitometry was used to
 evaluate band intensity. 6\. Lung Tissue Metastatic Lesions  Lung
 metastatic lesions were quantified from Hematoxylin and Eosin (H&E)
 stained lung tissue. 7\. Echocardiography data Transthoracic
 echocardiography was performed on all animals before and after Dox
 treatments  using a Vevo 2100 LAZR ultrasound system in M-mode. Shown are
 HR (heart rate), Ds (Systolic diameter), Dd (Diastolic diameter), Vs
 (Systolic volume), Vd (Diastolic volume), SV (Stroke volume), EF (Ejection
 fraction), FS (Fractional shortening), MVE (Mitral valve E-wave), IVRT
 (Isovolumetric relaxation time), RR (Interval between R wave and R wave 
 in the EKG), E’ (Peak velocity of early diastolic transmitral flow) and
 E/E’ (ratio of E to Peak velocity of early diastolic mitral annular motion
 as determined by pulsed wave Doppler). In this table data are presented
 for each measurement of each animal under each treatment; below each group
 is the statistical summary (mean, standard deviation (SD), number of
 animals (N), and standard error (SE)). At the bottom, the results of the
 two treatment groups are analyzed using a nonpaired, two-sided Student’s
 t-test, reported as p values, for differences before and after the
 experimental treatments. ## Code/software None