This inconsistency could be due to the fact that studies were performed in a different cell line where HL-1 cells are cultured in a medium containing norepinephrine. Norepinephrine is known to increase the metabolism of cardiomyocytes, which could be responsible for higher basal oxygen consumption compared to other in vitro models. DNA damage induced by the anthracyclines is thought to result from direct interaction with DNA, inhibition of the enzymes involved in DNA replication and repair and/or by inducing indirect damage through ROS. Damage to DNA was assessed by measuring the number of cH2AX positive foci present in the nuclei of HL-1 cells, where cH2AX is a marker of DNA double strand breaks associated with phosphorylation of the histone H2AX on the serine 139. Two hours after drug exposure, both doxorubicin and epirubicin induced high and relatively equal degrees of DNA damage based on the average number of cH2AX positive foci per nucleus. Consistent with the ROS data, non-pegylated liposomal-doxorubicin induced less DNA damage compared to doxorubicin and epirubicin. Finally we investigated the induction of apoptotic AbMole Halothane markers since anthracyclines, ROS generation and DNA damage induce apoptotic cell death. Caspase-3 is one of the executioner caspases found to be activated in the anthracycline model of cardiotoxicity as a consequence of dysfunctional mitochondria producing ROS, DNA damage or sarcoplasmic reticulum stress. The analysis of the cleavage of caspase-3, reflecting its activation, as well as measurement of the cleavage of a substrate common to caspase-3 and caspase-7, showed increases in the anthracyclines treated groups that paralleled the ROS levels. These findings were confirmed by analysis of Annexin V, another accepted marker of apoptosis. In all the three experimental approaches to determine apoptosis activation we found that non-pegylated liposomaldoxorubicin possesses a lower cardiotoxic profile. Doxorubicinol and epirubicinol are toxic secondary metabolites produced from doxorubicin and epirubicin, respectively. A limitation of this study is the lack of measurements of these metabolites, as the different treatments we used could have led to different levels of doxorubicinol or epirubicinol both in vivo and in vitro. However, this does not change the central findings here of the paper as we used the same doses of the drugs in different experimental setting. Furthermore, as it could be argued that the toxicity of the three anthracyclines in the cardiomyocyte study might reflect their relative antitumor activities, drug sensitivity was compared in MCF-7 breast tumor cells. These studies demonstrated that the three anthracyclines had similar capabilities to suppress breast tumor cell growth in vitro. Direct comparisons between epirubicin and liposomal doxorubicin are limited to one small study that included 160 patients with metastatic breast cancer who were followed for approximately 3 years which showed similar cardiac toxicity between the two drugs and the LITE study, another recently completed clinical trial in patients with breast cancer randomized to a non-pegylated liposomal-doxorubicin-based or epirubicin-based chemotherapy regimen to determine the incidence of clinical and subclinical cardiotoxicity using Tissue Doppler Analysis. Although limited by the small number of subjects and small changes in LV function, the LITE study suggests a superiority of the non-pegylated liposomal-doxorubicin-based approach.