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Unformatted text preview: [CANCER RESEARCH 62, 4592 4598, August 15, 2002] Doxorubicin Treatment in Vivo Causes Cytochrome c Release and Cardiomyocyte Apoptosis, As Well As Increased Mitochondrial Efficiency, Superoxide Dismutase Activity, and Bcl-2:Bax Ratio 1 April C. Childs, Sharon L. Phaneuf, Amie J. Dirks, Tracey Phillips, and Christiaan Leeuwenburgh 2 University of Florida, Biochemistry of Aging Laboratory, College of Health and Human Performance, Gainesville, Florida 32611 ABSTRACT There have been very few investigations as to whether mitochondrial- mediated apoptosis in vivo is the underlying mechanism of doxorubicin cardiotoxicity. Moreover, no investigations have been conducted to deter- mine whether there are adaptive responses after doxorubicin treatment. We administered a single dose of doxorubicin (20 mg/kg) to male rats and isolated intact mitochondria from their hearts 4 days later. Apoptosis, as determined by the amount of cytosolic mononucleosomal and oligonucleo- somal DNA fragments (180 bp or multiples), was significantly increased after doxorubicin treatment. In contrast, Troponin-T, a cardiac-specific marker for necrotic damage, was unaltered 4 days after doxorubicin treatment. Cytosolic cytochrome c increased 2-fold in the doxorubicin- treated rats and was significantly correlated ( r 5 0.88; P < 0.01) with the increase in caspase-3 activity observed. Moreover, the level of bleomyocin- detectable iron in serum was significantly increased and may have con- tributed to the increase in oxidative stress, which was indicated by an increase in cytosolic 8-iso prostaglandin F 2 a . Cytosolic copper zinc super- oxide dismutase activity also increased significantly further supporting the notion that doxorubicin increases superoxide radical production. In addition to adaptations to antioxidant defenses, other adaptive mecha- nisms occurred in the mitochondria such as an increase in the respiratory P/O ratio and an increase in the Bcl-2:Bax ratio. These findings demon- strate that doxorubicin induces oxidative stress and mitochondrial-medi- ated apoptosis, as well as adaptive responses by the mitochondria to protect cardiac myocytes in vivo . INTRODUCTION Doxorubicin is a powerful anthracycline antibiotic used to treat many human neoplasms, including acute leukemias, lymphomas, stomach, breast and ovarian cancers, Kaposis Sarcoma, and bone tumors (1). Doxorubicin may also cause cardiotoxicity when used for a prolonged period of time, thereby limiting its clinical use (2). The chronic side effects are irreversible and include the development of cardiomyopathy and ultimately congestive heart failure. Although recent evidence shows that less toxic doses of doxorubicin can be used effectively, heart failure in doxorubicin-treated patients can go unde- tected between 4 and 20 years after treatment cessation, causing some cancer patients to be unwilling to use doxorubicin (3). Therefore, it is essential to identify the mechanisms by which doxorubicin is cardio-...
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