Comparing Flaxseed and Perindopril in the Prevention of Doxorubicin and Trastuzumab-Induced Cardiotoxicity in C57Bl/6 Mice

Round 1

Reviewer 1 Report

The authors made an effort to address the concerns outlined in the initial review. The manuscript is certainly improved. However, some points could be better clarified by the authors.

1. In section Methods, the western blotting topic needs to be clarified.

• Page 4, lanes 159 and 160. Can explain the quantification using Coomassie blue staining? Did Coomassie blue staining was used as a loading control staining method in western blotting analysis?
• I continue without understanding what method was used to quantify the concentration of total protein. Lowry method? Bradford method? Please, introduce this information.
• Finally, I would like to see the whole membranes of each western blotting realized. Please introduce all whole membranes in supplementary data and not a representative blot of NF-kB.

Author Response

1. The authors made an effort to address the concerns outlined in the initial review. The manuscript is certainly improved. However, some points could be better clarified by the authors.

We thank the reviewer for their comment.

2. In section Methods, the western blotting topic needs to be clarified. Page 4, lines 159 and 160. Can explain the quantification using Coomassie blue staining? Did Coomassie blue staining was used as a loading control staining method in western blotting analysis? I continue without understanding what method was used to quantify the concentration of total protein. Lowry method? Bradford method? Please, introduce this information.

We thank the reviewer for their query. In the revised manuscript, the following statement had been added on page 4, lines 160-162: “Total protein extraction from heart tissue was performed on a subset of mice from each group and subsequently quantified by use of the Bradford assay, as previously described.”

3. Finally, I would like to see the whole membranes of each western blotting realized. Please introduce all whole membranes in supplementary data and not a representative blot of NF-kB.

We thank the reviewer for their suggestion. As recommended, we have now included the entire membrane as supplementary data.

Reviewer 2 Report

In this current study, “Comparing flaxseed and perindopril in the prevention of doxorubicin and trastuzumab-induced cardiotoxicity in C57Bl/6 mice”, the authors test the efficacy of nutraceutical agent flaxseed on the prevention of DOX+ TRZ caused cardiotoxicity in breast cancer patients. They use the C57Bl/6 female mice as subjects, and gave mice a six-week treatment to evaluate the drug effects. At the end, they found the flaxseed is comparable with PER in the prevention of DOX+TRZ-mediated cardiotoxicity in this murine model. This study is less of novelty as that similar studies have been carried to evaluate different drug effects in terms of reducing the adverse cardiotoxicity side effect of chemotherapy drugs. But this paper with very solid results showed the importance of FLX as a potential preventive/therapy agent for the protective effect of chemotherapy drugs induced cardiotoxicity. It could be of interest for a particular group of readers. Please describe your study design and experimental procedure with more information.

Author Response

1. In this current study, “Comparing flaxseed and perindopril in the prevention of doxorubicin and trastuzumab-induced cardiotoxicity in C57Bl/6 mice”, the authors test the efficacy of nutraceutical agent flaxseed on the prevention of DOX+ TRZ caused cardiotoxicity in breast cancer patients. They use the C57Bl/6 female mice as subjects and gave mice a six-week treatment to evaluate the drug effects. At the end, they found the flaxseed is comparable with PER in the prevention of DOX+TRZ-mediated cardiotoxicity in this murine model. This study is less of novelty as that similar studies have been carried to evaluate different drug effects in terms of reducing the adverse cardiotoxicity side effect of chemotherapy drugs. But this paper with very solid results showed the importance of FLX as a potential preventive/therapy agent for the protective effect of chemotherapy drugs induced cardiotoxicity. It could be of interest for a particular group of readers. Please describe your study design and experimental procedure with more information.

We thank the reviewer for their comment. As suggested, we have included additional information related to the study design and experimental procedures. In the revised manuscript, we have updated the methods section on pages 2 and 3, lines 86-117 as follows:

“Animal procedures were executed in agreement with the guidelines of the Canadian Council on Animal Care. All procedures were approved by the Animal Protocol Review Committee at the University of Manitoba [REB: 17-022/3 (AC11285)]. The study design consisted of a six week study, composed of a three week prophylactic period and a three week treatment period. A  total of 81 wild-type C57Bl/6 female mice (8-12 weeks old; Jackson Laboratories, Bar Harbor, ME, US) were randomly assigned to receive either regular chow (RC) (no. 9GR2, Prolab RMH 3000; TestDiet), 10% FLX-supplemented diets (no. 9GR0, modified Prolab RMH 3000 grain-based semipurified, TestDiet), and/or PER (3 mg/kg) daily for a total of 6 weeks (Figure 1). FLX-supplemented study diets were prepared by TestDiet manufacturing, as previously described [8]. Briefly, 10% FLX was added to modified rodent LabDiet Rat/Mouse/Hamster 3000 with fat sources removed. Diets were assessed and changed for each animal (5 g pellet diet/day) once per week.

After the 3-week prophylactic period, mice were randomized to receive 3 weekly intraperitoneal injections of: i) 0.9% Saline; or ii) DOX (8 mg/kg/week) + TRZ (3mg/kg/week) to produce a murine model of chronic cardiac dysfunction caused by chemotherapy (Figure 1) [5,6,8,9,24]. The 5 main study groups included RC + Saline (n=16), RC + DOX + TRZ (n=20), FLX + DOX + TRZ (n=18), PER + DOX + TRZ (n=10), and FLX + PER + DOX + TRZ (n=17). The administration schedule of DOX (24 mg/kg; Doxorubicin, Pfizer), TRZ (9 mg/kg; Herceptin, Hoffman-La Roche), and PER (9 mg/kg; Coversyl, Servier) in our chronic in vivo murine model was to recapitulate the dosage used in the clinical setting that has been validated by our group and others [8, 24].

Throughout the 6-week study, mice had ad libitum access to water and RC and 10% FLX-supplemented diets. All animals were maintained on a 12-hour day/night cycle throughout the 6-week period. At the end of the six weeks, mice were euthanized by an intraperitoneal injection of 110 mg/kg of pentobarbital buffered with 2% lidocaine. At study end point, cardiac tissue was used for histological and biochemical analyses including for Western blot analysis. In addition, blood samples were obtained for plasma oxylipin analysis. Plasma was prepared by adding 300 mL of cardiac blood into a Microvette CB 300 300 tube coated with K2EDTA anticoagulant (Cat. # NC9141704: SARSTEDT Inc.) and centrifuged at 2000 relative centrifugal force for 5 minutes. Plasma was then collected and stored at -80 degrees Celsius until analyses.”