Year of Publication

2015

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Toxicology and Cancer Biology

First Advisor

Dr. Mary Vore

Second Advisor

Dr. Daret St Clair

Abstract

Doxorubicin (DOX) is a broad-spectrum and effective chemotherapeutic agent, but its use in oncologic practice is limited by dose-dependent cumulative cardiotoxicity. DOX-induced cardiotoxicity is in large part due to its ability to cause oxidative stress. Multidrug resistance associated protein 1 (MRP1/ABCC1) is a member of the ATP-binding cassette (ABC) transporter superfamily. By effluxing a wide variety of endogenous and exogenous substrates, Mrp1 plays important physiological roles in multiple tissues and also protects normal tissues against toxicants. However, the role of MRP1 in heart is largely unknown.

The role of Mrp1 in DOX-induced cardiotoxicity was investigated in Mrp1 null (Mrp1-/-) and their C57BL (WT) littermates. Chronic DOX caused body weight loss and hemotoxicity, and these adverse effects were significantly exacerbated in Mrp1-/- vs WT mice. Importantly, loss of Mrp1 potentiated DOX-induced cardiotoxicity, presenting as worsened cardiac function and more cellular apoptosis in DOX treated Mrp1-/- mice. Mrp1 also protected neonatal mouse cardiomyocytes (CM) and cardiac fibroblasts (CF) culture against DOX cytotoxicity in vitro. This was demonstrated by the decreased cell survival, more apoptosis and more DNA damage in DOX treated Mrp1-/- vs WT cells.

In addition, the effects of deletion of Mrp1 was studied on glutathione (GSH)/glutathione disulfide (GSSG) homeostasis, glutathione conjugate of 4-hydroxy-2-nonenal (GS-HNE) accumulation, protein oxidative damage and expression of antioxidant enzymes. Loss of Mrp1 led to significantly higher GSH and GSSG basal levels in heart. Following DOX treatment, Mrp1-/- CM and CF showed increased GSH and GSSG levels vs WT cells. Meanwhile, DOX increased expression of the GSH synthesis enzymes in Mrp1-/- but not WT cells. Thus, increased GSH synthesis may contribute to the further increase in the GSH pool in DOX-treated Mrp1-/- cells. DOX induced comparable increases of GS-HNE concentration in WT and Mrp1-/- mice hearts. Finally, expression of extracellular superoxide dismutase (ECSOD/SOD3) was significantly lower in Mrp1-/- vs. WT CM treated with either saline or DOX.

In summary, this study is the first to document a protective role of Mrp1 in DOX-induced cardiotoxicity. It gives critical information regarding the potential adverse sequelae of introduction of MRP1 inhibitors as adjuncts to clinical chemotherapy of multidrug resistant tumors.

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