Dataset DOI: 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 × 10⁶ 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 mm³, 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 (NO₃^-) and nitrite (NO₂^-) 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