Protection against Doxorubicin-Induced Cardiotoxicity by Ergothioneine

Figures

• 

  Figure A1. Image of excised tumours from each animal (animals euthanized at differing points corresponding to images (A–C)). Allocation of groups can be found in the table.

• 

  Figure 1. Timeline of animal studies and groups. (A) Doxorubicin cardiotoxicity animal study in C57BL6 mice with or without ET administration. Mice (except for saline controls) were given 6 mg/kg doxorubicin weekly for 3 weeks and monitored periodically for a further 5 weeks. (B) A breast cancer model using injection of 4T1 cells into the mammary fat pad of BALB-C mice was used to evaluate the direct effect of ET on tumor growth and doxorubicin chemotherapy. Animals administered with doxorubicin were given 6 mg/kg doxorubicin weekly for 3 weeks. In both studies, animals supplemented with ET were given 70 mg/kg ET daily for 7 days prior to the commencement of the study, then once weekly thereafter until the completion of the study.

• 

  Figure 2. Animal weights. Animal weights were taken at each timepoint to compare weights of animals with doxorubicin administration with (doxo + ET) and without (doxo) ET supplementation with untreated (saline control) animals. Mice administered with doxorubicin had a significant decline in mean body weight compared with saline controls. Doxorubicin-treated animals supplemented with ET also had significant declines in body weight; however, their body weights were able to recover, being significantly greater than doxorubicin-treated animals alone by week 8. A 2-way analysis of variance (ANOVA) using multiple column comparison was performed with significances of **** p < 0.0001, *** p < 0.001 vs. saline control, and # p < 0.05 vs. doxorubicin alone.

• 

  Figure 3. Assessment of cardiac function. (A) The change in LVEF was measured as an indicator of cardiac (systolic) function. Significant decreases in LVEF were seen with doxorubicin-alone-administered animals, which was significant versus control from 2 weeks. Supplementation of doxorubicin-treated animals with ET prevented this decline in LVEF, which was significantly different to doxorubicin-alone-treated animals at the 3-week time point. (B) Similarly, myocardial performance index was significantly increased in doxorubicin-treated animals (indicating declining cardiac function). ET administration decreased MPI but not to baseline/saline control levels. A 2-way ANOVA using multiple column comparison was performed, with indicated significance levels; **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05 vs. saline control, and #### p < 0.0001, ### p < 0.001, ## p < 0.01 vs. doxorubicin alone.

• 

  Figure 4. Levels of ergothioneine and hercynine in the heart. (A) Real-time PCR analysis of OCTN1 (the ET transporter) mRNA expression in heart tissue. Although a slight increase in OCTN1 mRNA was seen with doxorubicin administration, this was not significant. No changes were seen in doxorubicin-treated animals with ET supplementation. (B) Significantly lower levels of ET were seen in the heart with doxorubicin treatment. This may be due to increased consumption of ET as it scavenges ROS. The heart levels of hercynine (C) also decreased significantly relative to saline control. As expected, supplementation with ET increased levels of hercynine (prior studies show that levels of hercynine correlated closely with ET [54]). A 1-way ANOVA with Tukey’s multiple comparison test was performed; ** p < 0.01, * p < 0.05 vs. saline control; p < 0.0001 saline control or doxorubicin alone vs. doxorubicin + ET groups in (B,C); ns—not significant.

• 

  Figure 5. Plasma ergothioneine and hercynine. A significant decrease was seen in plasma (A) ET levels with administration of doxorubicin relative to saline controls, similar to what was observed in the heart. A non-significant increasing trend for plasma hercynine was observed following doxorubicin administration (B). As expected, supplementation increased plasma levels of ET and hercynine. A 1-way ANOVA with Tukey’s multiple comparison test was performed; (A) * p < 0.05, **** p < 0.0001 vs. saline controls; (B) * p < 0.05 vs. doxorubicin alone.

• 

  Figure 6. Oxidative damage biomarkers. Although slight increases were observed in the levels of 8OHdG, a biomarker of oxidative DNA damage (A), and 8OHG, a biomarker of oxidative RNA damage (B), with doxorubicin administration relative to saline controls, this was not significant (ns; 1-way ANOVA, Tukey’s multiple comparison test). Supplementation with ET also appeared to decrease oxidative markers of both DNA and RNA, but this was not significant (A,B). No differences were seen in heart protein carbonyls (C) between any of the groups. A similar trend in plasma protein carbonyls (D) was seen as with nucleic acid oxidation markers in heart, but again this was not significantly different.

• 

  Figure 7. Breast cancer model. (A) Administration of doxorubicin resulted in a significant (p < 0.0001; 1-way ANOVA multiple comparison test) decline in body weights with or without ET supplementation relative to saline controls. ET-supplemented-animal weights remained similar to saline controls. (B) Cardiac function (LVEF) was measured at baseline and endpoint in the breast cancer model. As with the C57BL-6J doxorubicin model, a significant decrease in LVEF was seen with administration of doxorubicin relative to saline control animals. However, supplementation with ET significantly prevented this decline in cardiac function (2-way ANOVA with multiple comparison test; ** p < 0.01 vs. saline control # p < 0.05 vs. doxo + ET). The average volume (C) (estimated based on caliper measurements; Vol(mm3) = [width(mm)]2 x [length(mm)/2]) and weight (D) of the excised tumors are shown. Saline control animals had the largest tumor size, but administration of doxorubicin significantly decreased volume/weight of the tumor (* p < 0.05; 1-way ANOVA with multiple comparison test). Supplementation of animals with ET alone had a slight, but not significant, decrease in tumor size compared with saline…