Blood plasma profile after DOX treatment.

#<p>For troponin I analysis, only 13 and 16 samples were measured in acute and sub-chronic protocol, respectively due to limitations in the analytical kit used.</p><p>Differences between treatment groups means within the same model were evaluated by Student’s t test, when assumptions were not achieved a Welch correction or the non-parametric Mann-Whitney test were applied (see Material and Methods for detailed information).</p>*<p>p≤0.05;</p>**<p>p≤0.01;</p>***<p>p≤0.001 vs saline group of the same model. List of abbreviations: CK – creatine kinase; TnI – troponin I; TRIG – triglycerides; CHOL – total cholesterol; AST – aspartate aminotransferase; ALT – alanine aminotransferase; TP – total serum proteins; LDH – lactate dehydrogenase; CREA – creatinine; UA – uric acid; BUN – blood urea nitrogen; SE – standard error.</p

Body and organs mass profile of animals subjected to DOX treatment protocols.

<p>Data refers to wet organ mass and its ratio to body mass was obtained dividing the organ mass over the respective total animal mass times 1000. The deceased DOX-treated rat and its matched control in the sub-chronic model were excluded from this analysis. Differences between treatment groups means within the same model were evaluated by Student’s t test (see Material and Methods for detailed information).</p>*<p>p≤0.05;</p>**<p>p≤0.01;</p>***<p>p≤0.001 vs saline group of the same treatment protocol. HM:BM – heart mass to body mass ratio; LM:BM – liver mass to body mass ratio; KM:BM – kidney mass to body mass ratio; SE – standard error.</p

Histological analysis of organs collected from rats treated with DOX.

<p>No notorious differences or hallmarks of DOX toxicity were found in the different tissues in both protocols. Panels represent HE photographs of random chosen tissues: hearts present minor cytoplasmatic vacuolization (Panel <b>B</b>) and cytoplasmatic dilatation (Panel <b>D</b>); liver usually show minor cytoplasmatic vacuolization (Panel <b>F</b> and <b>H</b>); no changes in kidneys (Panel <b>J</b> and <b>L</b>). Organs were fixed in Bouin’s solution, processed through standard histological procedures and stained with HE (for more information, see Material and Methods).</p

DOX decreases body mass gain over time in a sub-chronic toxicity model.

<p>After the fourth injection, the body mass of DOX-treated animals started to be distinctly different from the saline-treated and therefore the growth profile is dramatically changed at the end of treatment. Only one DOX-treated animal died during the protocol (indicated by the black arrow). Animals in the control group are depicted by open circles while DOX-treated animals are in full circles. Lines represent the means of each group at each time point. S – sacrifice time-point.</p

Treatment with DOX markedly affects mitochondrial respiration during ADP phosphorylation.

<p>The effect is clearly observed in heart mitochondria during sub-chronic treatment but absent when animals were acutely-treated. A reverse pattern is observed in the other two organs tested. Data represents mitochondrial oxygen consumption rates measured with a Clark electrode (for details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038867#s2" target="_blank">Materials and Methods</a>) where 225–250 nmol ADP was added to induce state 3 respiration. State 4 is generally described as the rate of oxygen consumption after complete phosphorylation of added ADP. <b>A</b> – heart; <b>B</b> – liver; <b>C</b> – kidney. Bars represent means of treatment groups (saline in <b>white bars</b>; DOX in <b>black bars</b>) with SE. Differences between treatment groups means within the same model were evaluated by matched pairs Student’s t test to exclude the variability related to mitochondrial isolation and electrode calibration but when assumptions were rejected the non-parametric Wilcoxon matched pairs test was applied (see Material and Methods for detailed information). *, p≤0.05; **, p≤0.01; ***, p≤0.001 vs saline group of the same model. n = 10, 9 and 10 (acute model – heart, liver and kidney, respectively) or n = 12, 11 and 12 (sub-chronic model – heart, liver and kidney, respectively). GM - glutamate/malate; SUC - succinate.</p

Echocardiogram parameters in the sub-chronic protocol.

<p>Differences between treatment groups were evaluated by non-parametric Mann-Whitney test due to their lack of normality (see Material and Methods for detailed information). IVS – interventricular septum; LPWT – left posterior wall thickness; LVDd – left ventricular diastolic dimension; LVDs – left ventricular systolic dimension; LVEF – left ventricular ejection fraction; FS – fraction shortening; AT s/d – arterial tension systole/diastole.</p

Cellular ultra-structure remains intact after acute or sub-chronic DOX treatment.

<p>No notorious differences or hallmarks of DOX toxicity were found in the different tissues in both protocols. Panels represent electron microphotographs of randomly chosen tissues: hearts present well-defined Z-bands and organized myofibrils (Panel <b>C</b>,<b>D</b> and <b>L</b>–<b>O</b>) and minor cytoplasmatic dilatation (Panel <b>M</b> and <b>O</b>); livers show cytoplasmatic vacuolization (Panel <b>E</b>, <b>G</b> and <b>S</b>) and lipid-like droplets structures (Panel <b>Q</b>) and some mitochondria appear in the condensed conformation (Panel <b>G</b>); renal mitochondria appear in an intermediated conformation between orthodox and condensed form (Panel <b>X</b>). Organs were fixed in 4% gluteraldehyde and post-fixed in osmium (for more information, see Material and Methods).</p

Effects of DOX on mitochondrial transmembrane electric potential.

<p>Data was collected with a TPP⁺-sensitive electrode (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038867#s2" target="_blank">Materials and Methods</a>) where 225–250 nmol ADP were added to induce depolarization (phosphorylative cycle). Differences between treatment groups means within the same model were evaluated by matched pairs Student’s t test to exclude the variability related to mitochondrial isolation and electrode calibration but when assumptions were rejected the non-parametric Wilcoxon matched pairs test was applied (see Material and Methods for detailed information).</p>*<p>p≤0.05;</p>**<p>p≤0.01 vs saline group of the same model. SE – standard error.</p

Respiratory Control Ratio (RCR) and ADP phosphorylated per consumed oxygen ratio (ADP/O).

<p><b>A</b> – heart; <b>B</b> – liver; <b>C</b> – kidney. Bars represent means of treatment groups (saline in <b>white bars</b>; DOX in <b>black bars</b>) with SE. Differences between treatment groups means within the same model were evaluated by matched pairs Student’s t test to exclude the variability related to mitochondrial isolation and electrode calibration but when assumptions were rejected the non-parametric Wilcoxon matched pairs test was applied (see Material and Methods for detailed information). *, p≤0.05 vs saline group of the same model. n = 10, 9 and 10 (acute model – heart, liver and kidney, respectively) or n = 12, 11 and 12 (sub-chronic model – heart, liver and kidney, respectively). GM - glutamate/malate; SUC - succinate.</p