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Reproductive hormone levels and differential mitochondria-related oxidative gene expression as potential mechanisms for gender differences in cardiosensitivity to Doxorubicin in tumor-bearing spontaneously hypertensive rats

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Abstract

Purpose

Chemotherapy with doxorubicin (Dox) causes dose-limiting cardiotoxicity. We investigated the role that gender has on cardiosensitivity to Dox treatment by evaluating reproductive hormone levels in male, castrated male (c-male), female and ovariectomized female (o-female) adult spontaneously hypertensive rats (SHRs) and expression of mitochondria-related genes in male and female adult SHRs.

Methods

SST-2 breast tumor-bearing SHRs were treated with saline, Dox, dexrazoxane (Drz) or both Dox and Drz and monitored for 14 days. Tumor size was used to monitor anticancer activity. Heart weight, cardiac lesion score and serum levels of cardiac troponin T (cTnT) were used to determine cardiotoxicity. Serum estradiol (E2) and testosterone were evaluated using electrochemiluminescence immunoassays. Expression of mitochondria-related genes was profiled in heart by MitoChip array analyses.

Results

Dox significantly reduced tumor volume (±Drz) and increased heart weight in all genders (13–30 % vs. control). Higher heart lesion scores were observed in reproductively normal animals (male 2.9, female 2.2) than in hormone-deficient animals (c-male 1.7, o-female 1.9). Lesion score and cTnT inversely correlated with hormone levels. Reduced levels of both sex hormones were observed after Dox treatment. Gene expression analyses of Dox-treated hearts showed significant differential expression of oxidative stress genes in male hearts and apoptotic genes in both male and female hearts.

Conclusions

Our results demonstrate that adult tumor-bearing male SHRs are more cardiosensitive to Dox than female or hormone-deficient animals. We provide evidence to suggest that reproductive hormones negatively regulate or are inhibited by Dox-induced cardiotoxicity and the selective cytotoxic mechanism likely functions through the greater activation of oxidative stress and apoptosis in male SHRs.

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Acknowledgments

We would like to acknowledge Eugene Herman (FDA/CDER) and Rodney Levine (NIH/NHLBI) for helpful discussions, and Karlecia Corbett and Raissa Dantas (FDA/CDER) for assistance with animal procedures. We gratefully acknowledge funding support by the United States Food and Drug Administration Office of Women’s Health grant. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the United States Food and Drug Administration and the Department of Health and Human Services, nor does the mention of trade names, commercial products or organizations imply endorsement by the United States Government.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

    Authors and Affiliations

    1. Laboratory of Chemistry, Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Ave/Bldg 52/72 Rm 2212, Silver Spring, MD, 20993, USA

      Yanira Gonzalez, Kaytee L. Pokrzywinski, Elliot T. Rosen, Baikuntha Aryal, Leena M. Chehab & V. Ashutosh Rao

    2. Center for Food Safety and Applied Nutrition, Food and Drug Administration, Silver Spring, MD, USA

      Steven Mog

    3. Personalized Medicine Branch, Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, 72079, AR, USA

      Vikrant Vijay, Carrie L. Moland & Varsha G. Desai

    4. Division of Molecular Genetics and Pathology, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, USA

      Jennifer S. Dickey

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    1. Yanira Gonzalez
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    Correspondence to V. Ashutosh Rao.

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    Yanira Gonzalez and Kaytee L. Pokrzywinski have contributed equally to this work.

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    Suppl Fig 1

    Change in body weight throughout 14-day drug treatment. SHR/SST-2s were separated by gender (n=20): male (A), c-male (B), female (C) and o-female (D) and administered either saline (circle), Dox [(10 mg/kg, iv), square], Drz [(50 mg/kg, ip) triangle] or a combination of Drz+Dox (downwards triangle) (n=5). Prior to the drug treatment, an initial body weight was taken for every animal. Body weights (1) were measured again after 4, 7, 11 and 14 days exposure to the corresponding treatment. Points denote the average percent change in weight, positive values indicate net growth and negative values indicate weight loss. Error bars represent one standard deviation from the mean. (EPS 18591 kb)

    Suppl Fig 2

    Change in tumor volume throughout 14-day drug treatment. SHR/SST-2s were separated by gender (n=20): male (A), c-male (B), female (C) and o-female (D) and administered either saline (circle), Dox [(10 mg/kg, iv), square], Drz [(50 mg/kg, ip) triangle] or a combination of Drz+Dox (downwards triangle) (n=5). Prior to the drug treatment, an initial body weight was taken for every animal. Tumor volumes (2) were recorded at days 7, 11 and 14. Points denote the average percent change in volume. Error bars represent one standard deviation from the mean. (EPS 17751 kb)

    Suppl Fig 3

    Cardiac mitochondria-related gene expression. Male (A) and female (B) SHR/SST-2 cardiac tissues were analyzed for changes in mitochondria-related gene expression in saline and Dox treatments. In male (A) and female (B) SHR/SST-2 hearts, 918 genes were analyzed. Genes that were not significantly expressed are light (circle) and genes in which expression significantly changed (p < 0.05) are dark (circle). Genes with greater than 2-fold changes in expression are dark square with overlaid light circle. Points represent the –log10 p-value vs the fold change in expression. Values above 1.0 indicate up-regulation, while values below 1.0 indicate down-regulation. (EPS 3933 kb)

    Suppl Fig 4

    Significantly up-regulated mitochondria-related genes in cardiac tissue. Each bar represents the –log10 (p-value) of each statistically significant individual gene (saline vs. Dox) based on gene ontology family for both male (blue) and female (pink) SHR/SST-2s. Specific genes are on the y-axis, while gene ontology families are to the left of the gene name. Gene families are in alphabetical order. If a gene belongs to more than one family, it is listed either to the right of the individual bar or to the left of the gene family. Statistical significance was determined at an alpha of 0.05. (EPS 5030 kb)

    Suppl Fig 5

    Significantly down-regulated mitochondria-related genes (A) and genes with greater than 2-fold change in expression (B) in cardiac tissue. For down-regulated genes (A), each bar represents the –log10 p-value of each statistically significant individual gene (saline vs. Dox), based on gene ontology family for both male (blue) and female (pink) SHR/SST-2s. Statistical significance was determined at an alpha of 0.05. For genes with greater than 2-fold change in expression (B), each bar depicts the fold change in expression between saline and Dox-treated male SHR/SST-2s (blue). For both panels, specific genes are on the y-axis, while gene ontology families are to the left of the gene name. Gene families are in alphabetical order. (EPS 4900 kb)

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    Gonzalez, Y., Pokrzywinski, K.L., Rosen, E.T. et al. Reproductive hormone levels and differential mitochondria-related oxidative gene expression as potential mechanisms for gender differences in cardiosensitivity to Doxorubicin in tumor-bearing spontaneously hypertensive rats. Cancer Chemother Pharmacol 76, 447–459 (2015). https://doi.org/10.1007/s00280-015-2786-8

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