Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis.
Generation of reactive oxygen species and mitochondrial dysfunction has been implicated in doxorubicin-induced cardiotoxicity. This study examined pro-apoptotic mitochondrial cell death signals in an H9C2 myocyte rat cell line and in isolated rat heart mitochondria exposed to doxorubicin. Mitochondrial and cellular viability were assessed using an MTT viability assay (formazan product formed by functional mitochondrial dehydrogenases) and calcein AM dye (fluoresces upon cleavage by cytosolic esterases). Mitochondrial dysfunction followed by cell death was observed using nM concentrations of doxorubicin. Significant doxorubicin-induced cell death was not apparent until after 6 h following doxorubicin exposure using the calcein AM assay. The involvement of apoptosis is evidenced by an increase in TUNEL (terminal (TdT)-mediated dUTP-biotin nick end labeling)-positive nuclei following doxorubicin treatment. Furthermore, doxorubicin administered to isolated mitochondria induced a rapid increase in superoxide production, which persisted for at least 1 h and was followed by increased cytochrome c efflux. In addition, caspase-3 activity was increased with doxorubicin administration in the H9C2 myocyte cell line. An oxidant-mediated threshold of mitochondrial death may be required for doxorubicin-induced apoptosis.
Green
Pattie S
PS
University of Florida, Biochemistry of Aging Laboratory, P.O. Box 118206, Gainesville, FL 32611, USA.
Leeuwenburgh
Christiaan
C
D000818:Animals; D017209:Apoptosis; D009202:Cardiomyopathies / Q000139:chemically induced / Q000517:prevention & control; D002460:Cell Line; D002470:Cell Survival; D003574:Cytochrome c Group / Q000032:analysis / Q000378:metabolism; D004317:Doxorubicin / Q000633:toxicity*; D005609:Free Radicals / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000502:physiology; D032342:Muscle Cells / Q000187:drug effects; D051381:Rats; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors
D003574:Cytochrome c Group; D005609:Free Radicals; D013481:Superoxides; D004317:Doxorubicin